Lipids and essential oils as antimicrobial agents

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Lipids and Essential Oils

Editor

Thormar

as Antimicrobial Agents

Lipids and Essential Oils as Antimicrobial Agents is a comprehensive review of the scientific knowledge in this field. International experts provide summaries on: • • • • • • • • • •

antibacterial, antiviral and antifungal activities of lipids membranes as targets of antimicrobial lipids antimicrobial lipids in milk antimicrobial lipids of the skin and tear fi lm antimicrobial lipids and innate immunity lipids as active ingredients in pharmaceuticals, cosmetics and health foods antimicrobial lipids as disinfectants, antiseptics and sanitizers the chemistry and bioactivity of essential oils antiviral effects of plant-derived essential oils and pure oil components antibacterial and antifungal activities of essential oils

Lipids and Essential Oils as Antimicrobial Agents is an essential guide to this important topic for researchers and advanced students in academia and research working in pharmaceutical, cosmetic and food sciences, biochemistry and natural products chemistry, microbiology; and for health care scientists and professionals working in the fields of public health and infectious diseases. It will also be of interest to anyone concerned about health issues and particularly to those who are conscious of the benefits of health food and natural products.

as Antimicrobial Agents

Lipids and essential oils have strong antimicrobial properties - they kill or inhibit the growth of microbes such as bacteria, fungi, and viruses. They are being studied for use in the prevention and treatment of infections, as potential disinfectants, and for their preservative and antimicrobial properties when formulated as pharmaceuticals, in food products, and in cosmetics.

Lipids and Essential Oils

Editor: Halldor Thormar Faculty of Life and Environmental Sciences, University of Iceland

Editor Halldor Thormar

Lipids and Essential Oils

as Antimicrobial Agents

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Lipids and Essential Oils as Antimicrobial Agents Editor

HALLDOR THORMAR Faculty of Life and Environmental Sciences, University of Iceland, Reykjavik, Iceland

A John Wiley and Sons, Ltd., Publication

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This edition first published 2011
C 2011 John Wiley & Sons, Ltd Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com. The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for every situation. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Lipids and essential oils as antimicrobial agents / editor, Halldor Thormar. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-74178-8 (cloth) – ISBN 978-0-470-97661-6 (ebook) – ISBN 978-0-470-97662-3 (obook) 1. Anti-infective agents. 2. Lipids–Therapeutic use. 3. Essences and essential oils–Therapeutic use. I. Thormar, Halldor. [DNLM: 1. Anti-Infective Agents. 2. Lipids–pharmacology. 3. Oils, Volatile–pharmacology. QV 250] RM267.L57 2011 615 .792–dc22 2010037014 A catalogue record for this book is available from the British Library. Print ISBN: 9780470741788 eBook ISBN: 9780470976616 oBook ISBN: 9780470976623 ePub ISBN: 9780470976678 Typeset in 10/12pt Times by Aptara Inc., New Delhi, India. Printed and bound in Singapore by Markono Print Media Pte Ltd.

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Contents

List of Contributors Introduction

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1

Membranes as Targets of Antimicrobial Lipids 1 Peter J. Quinn 1.1 Introduction 2 1.2 Oil and Water Don’t Mix! 2 1.3 Polar Lipids 3 1.3.1 The Amphiphilic Character of Polar Lipids 3 1.3.2 Hydrophobic Constituents of Lipids 4 1.3.3 Polar Groups of Complex Lipids 5 1.4 Properties of Surfactants 7 1.4.1 Critical Micelle Concentration 7 1.4.2 Aggregation of Surface-Active Molecules 8 1.4.3 The Influence of Solvent 9 1.5 Cell Membranes 11 1.5.1 Membrane Lipids 11 1.5.2 Lipid Domains in Membranes 13 1.5.3 Membrane Proteins 15 1.5.4 Membrane Stability 15 1.5.4.1 Membrane Lipid Phase Behaviour 17 1.5.4.2 Membrane Lipid Homeostasis in Homoiothermic Organisms 18 1.5.4.3 Membrane Lipid Adaption in Poikilothermic Organisms 19 1.6 The Action of Antimicrobial Lipids on Cell Membranes 20 1.7 Conclusions 21 Acknowledgements 21 References 22

2

Antibacterial Effects of Lipids: Historical Review (1881 to 1960) Halldor Thormar 2.1 Introduction 2.2 Antibacterial Activity of Soaps 2.2.1 Antibacterial Action of Fatty Acids and Their Derivatives 2.3 Inhibition of Lipids and Serum Albumin against the Antibacterial Action of Soaps 2.4 Diverse Actions of Fatty Acids and Their Salts on Bacteria

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The Nature of the Bactericidal Action of Fatty Acids A Possible Role of Soaps and Fatty Acids in Host Defence against Bacteria Studies of Prophylactic and Therapeutic Applications of Soaps and Fatty Acids Conclusions References

3 Antibacterial, Antiviral and Antifungal Activities of Lipids Gudmundur Bergsson, Hilmar Hilmarsson and Halldor Thormar 3.1 Introduction 3.2 Antibacterial Activities of Fatty Acids and Monoglycerides 3.2.1 Activities against Gram-Positive Bacteria In Vitro 3.2.2 Activities against Gram-Negative Bacteria In Vitro 3.2.3 Antibacterial Mechanism 3.2.4 A Possible Role of Antimicrobial Lipids in Host Defence against Bacteria 3.3 Antiviral Activities of Fatty Alcohols, Fatty Acids and Monoglycerides 3.3.1 Early Studies of Antiviral Lipids 3.3.2 Antiviral Activities of Milk Lipids 3.3.3 Antiviral Activities of Lipids and Fatty Alcohols 3.3.4 Antiviral Mechanism of Lipids 3.4 Antifungal Activities of Fatty Acids and Monoglycerides 3.4.1 In Vitro Studies 3.4.2 In Vivo Studies 3.5 Conclusions References 4 Antimicrobial Lipids in Milk Charles E. Isaacs 4.1 Introduction 4.2 Occurrence 4.2.1 Biosynthesis 4.2.2 Quantitative Assays 4.2.2.1 Measurement of Antibacterial Activity 4.2.2.2 Measurement of Antiviral Activity 4.3 Molecular Properties 4.3.1 Lipids in Milk and Plant Oils 4.3.2 Dependence of Antimicrobial Activity on Chain Length, Saturation and pH 4.4 Antimicrobial Activity 4.4.1 Mechanism of Action 4.4.2 Synergy between Antimicrobial Lipids and Antimicrobial Peptides 4.4.2.1 Antimicrobial Peptides and Proteins

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4.4.2.2

4.5

4.6 4.7

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Potential Synergism between Antimicrobial Milk Lipids and Peptides 4.4.3 Influencing Factors 4.4.4 Spectrum Applications 4.5.1 Additive Advantage to Foods and Biological Products 4.5.2 Physiological Advantage 4.5.3 Sexually Transmitted Pathogens and Topical Activity Safety, Tolerance and Efficacy Conclusions References

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Antimicrobial Lipids of the Skin and Tear Film Carol L. Bratt, Phil Wertz, David Drake, Deborah V. Dawson and Kim A. Brogden 5.1 Introduction 5.2 Innate Immune Mechanisms in Skin 5.2.1 The Extensive Cutaneous Microbial Burden 5.2.2 Cutaneous Innate Immune Mechanisms 5.3 Types and Locations of Lipids of the Skin and Tear Film 5.3.1 Hypodermal and Dermal Lipids 5.3.2 Epidermal Lipids 5.3.3 Secreted Lipids 5.3.4 Tear-Film Lipids 5.4 Functions of Lipids 5.4.1 Cutaneous Lipids as Permeability Barriers 5.4.2 Cutaneous Lipids as Innate Immune Mechanisms 5.5 Antimicrobial Activity of Lipids and Their Mechanisms of Killing 5.5.1 Lipid Deficiencies 5.5.2 Lipid Antimicrobial Activity and Mechanism of Action 5.5.2.1 Sphingolipids 5.5.2.2 Fatty acids 5.6 Synergy of Cutaneous Lipids and Other Innate Immune Molecules 5.7 Lipids as Therapeutic Agents 5.8 Conclusions Acknowledgements References Antimicrobial Lipids and Innate Immunity Halldor Thormar 6.1 Introduction 6.2 The Role of Human Milk Lipids in Innate Immunity 6.2.1 Breast-Feeding Protects Infants against Infection 6.2.2 Factors in Human Milk which Protect against Infection 6.2.3 Antmicrobial Lipids in Human Milk and Their Possible Protective Function

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6.3

Antimicrobial Lipids in the Pulmonary Mucosa 6.3.1 The Innate Immune System of the Respiratory Tract 6.3.2 Antimicrobial Fatty Acids in the Alveoli of Animal and Human Lungs 6.4 Antimicrobial Skin Lipids 6.4.1 The Self-Disinfecting Power of the Human Skin 6.4.2 The Role of Skin Lipids in the Self-Disinfection of Human Skin 6.4.3 Sebum Lipids as a Source of Antimicrobial Fatty Acids in the Skin 6.4.4 Stratum Corneum as a Source of Antimicrobial Lipids in the Skin 6.4.5 Antimicrobial Lipids as a Part of the Innate Immune Defence 6.5 Conclusions References

7 Lipids as Active Ingredients in Pharmaceuticals, Cosmetics and Health Foods Th´ord´ıs Kristmundsd´ottir and Sk´uli Sk´ulason 7.1 Introduction 7.2 Antimicrobial Effects of Lipids 7.2.1 Antibacterial Activity 7.2.2 Antiviral Activity 7.2.3 Antifungal Activity 7.3 Lipids in Pharmaceuticals 7.3.1 Lipids in Pharmacopeias 7.3.2 Microbicidal Lipids for Treatment of Mucosal and Skin Infections 7.3.3 Lipids as Penetration Enhancers in Pharmaceutical Dosage Forms 7.4 Microbicidal Lipids in Agriculture and Aquaculture 7.5 Lipids in Therapy 7.5.1 Effect of Lipids on Infectious and Inflammatory Diseases 7.5.2 Effect of Lipids on Psoriasis 7.5.3 Effect of Lipids on Cardiovascular Diseases 7.5.4 Effect of Lipids on Cognitive Function 7.6 Lipids in Cosmetics 7.6.1 Sources of Lipids Used in Cosmetics 7.6.2 Antimicrobial Activity of Lipids in Cosmetics 7.6.3 Other Activities of Lipids in Cosmetics 7.7 Lipids in Health Food 7.8 Conclusions References 8 Antimicrobial Lipids as Disinfectants, Antiseptics and Sanitizers Halldor Thormar and Hilmar Hilmarsson 8.1 Introduction 8.2 Soaps as Disinfectants and Antiseptics 8.3 Use of Bactericidal Lipids to Reduce Microbial Contamination of Food Products

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8.4

8.5

9

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Killing of Foodborne Bacteria by Glycerol Monocaprate (Monocaprin) Emulsions 8.4.1 Reduction of Viable Campylobacter in Drinking Water and Feed of Chickens by Addition of Monocaprin Emulsions 8.4.2 Reduction of Viable Campylobacter on Poultry Carcasses 8.4.3 Reduction of Psychrotrophic Spoilage Bacteria on Chicken Carcasses 8.4.4 Sanitation of Kitchen Surfaces by Washing with Monocaprin Emulsions Conclusions References

Chemistry and Bioactivity of Essential Oils Christine F. Carson and Katherine A. Hammer 9.1 Introduction 9.2 Chemistry of Essential Oils 9.2.1 Terpenes 9.2.1.1 Biosynthesis of Terpenes 9.2.1.2 Monoterpenes 9.2.1.3 Sesquiterpenes 9.2.1.4 Diterpenes 9.2.1.5 Norterpenes 9.2.2 Phenylpropanoids 9.2.2.1 Biosynthesis of Phenylpropanoids 9.2.2.2 Phenylpropanoids in Essential Oils 9.2.3 Sulfur and Nitrogen Compounds of Essential Oils 9.3 Biological Activity of Essential Oils 9.3.1 General Overview 9.3.2 Antimicrobial Activity 9.3.2.1 Antibacterial Activity 9.3.2.2 Antifungal Activity 9.3.2.3 Mechanisms of Antibacterial and Antifungal Action 9.3.2.4 Antiviral Activity 9.3.2.5 Antiprotozoal Activity 9.3.3 Anticancer Activity 9.4 Uses of Essential Oils 9.4.1 Pharmaceutical Products 9.4.2 Foods and Beverages 9.5 Conclusions References Antiviral Effects of Plant-Derived Essential Oils and Pure Oil Components Paul Schnitzler, Akram Astani and J¨urgen Reichling 10.1 Introduction 10.2 Characterization and Medicinal Use of Essential Oils

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Cytotoxicity of Essential Oils Antiviral Activities of Essential oils 10.4.1 In Vitro and In Vivo Studies of Antiherpesvirus Activity of Essential Oils 10.4.2 In Vitro and In Vivo Studies of Antiherpesvirus Activity of Pure Essential Oil Components 10.4.3 In Vitro and In Vivo Studies of Antiviral Effects of Essential Oils against Other Viruses 10.5 Mode of Antiviral Action of Essential Oils 10.6 Conclusions References

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Antibacterial and Antifungal Activities of Essential Oils Katherine A. Hammer and Christine F. Carson 11.1 Introduction 11.2 Methods for Quantifying Antimicrobial Activity 11.3 Antibacterial and Antifungal Essential Oils by Plant Family 11.3.1 Apiaceae 11.3.2 Lamiaceae 11.3.3 Lauraceae 11.3.4 Myrtaceae 11.3.5 Poaceae 11.3.6 Rutaceae 11.3.7 Other 11.3.8 Comparative Activities of Essential Oils 11.4 Antimicrobial Essential-Oil Components 11.4.1 Aldehydes 11.4.2 Alcohols 11.4.3 Phenols 11.4.4 Ketones 11.4.5 Esters 11.4.6 Oxides and Epoxides 11.4.7 Methyl Ethers 11.4.8 Terpene Hydrocarbons 11.4.9 Generalizations about Component Activity 11.5 Factors Influencing Activity 11.5.1 Microorganism-Related Factors 11.5.2 Environmental Conditions 11.6 Mechanisms of Action 11.6.1 Membrane and Membrane-Related Actions 11.6.2 Adaptation and Tolerance 11.6.2.1 Resistance 11.6.3 Other Antimicrobial Effects 11.6.3.1 Toxins, Enzymes and Virulence Factors 11.6.3.2 Biofilms and Quorum Sensing

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11.6.3.3

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11.8

11.9

Index

Synergy between Essential Oils or with Other Compounds 11.6.3.4 Other Clinical Efficacy of Essential Oils and Components 11.7.1 Animal Studies 11.7.2 Human Studies Toxicity of Essential Oils 11.8.1 In Vitro Toxicity Assays 11.8.2 Animal Studies 11.8.3 Toxic Effects in Humans Conclusions References

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List of Contributors

Akram Astani Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany Gudmundur Bergsson Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland Carol L. Bratt Dows Institute for Dental Research, College of Dentistry, The University of Iowa, Iowa City, IA, USA Kim A. Brogden Dows Institute for Dental Research, and The Department of Periodontics, College of Dentistry, The University of Iowa, Iowa City, IA, USA Christine F. Carson Discipline of Microbiology and Immunology (M502), School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia Deborah V. Dawson Dows Institute for Dental Research, and The Department of Preventive and Community Dentistry, College of Dentistry, The University of Iowa, Iowa City, IA, USA David Drake Dows Institute for Dental Research, and The Department of Endodontics, College of Dentistry, The University of Iowa, Iowa City, IA, USA Katherine A. Hammer Discipline of Microbiology and Immunology (M502), School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia Hilmar Hilmarsson Faculty of Life and Environmental Sciences, University of Iceland, Reykjavik, Iceland Charles E. Isaacs Department of Developmental Biochemistry, New York State Institute for Basic Research in Developmental Disabilities, NY, USA Th´ord´ıs Kristmundsd´ottir Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland

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List of Contributors

Peter J. Quinn Department of Biochemistry, King’s College London, London, United Kingdom ¨ Jurgen Reichling Department of Biology, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany Paul Schnitzler Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany ´ Skulason ´ Skuli Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland Halldor Thormar Faculty of Life and Environmental Sciences, University of Iceland, Reykjavik, Iceland Phil Wertz Dows Institute for Dental Research, and The Department of Oral Pathology, Radiology & Medicine, College of Dentistry, The University of Iowa, Iowa City, IA, USA

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Introduction

There has recently been a renewed interest in the antimicrobial effects of natural compounds which were commonly used as health remedies in the Western world until the advent of antibiotic drugs in the 1940s and 50s. After the emergence of antibiotics many previously fatal infections and infectious diseases were brought under control and millions of lives were saved. Due to the dramatic effect of the new synthetic drugs, some health professionals even believed that the threat to mankind of pathogenic microorganisms had finally been eliminated. The great success of chemotherapy, using synthetic antibiotics against bacterial and fungal infections and nucleoside analogues against viral infections, discouraged researchers and the pharmaceutical industry from making serious efforts to develop drugs containing simple natural compounds. However, this may now be changing, with the increasing problem of drug-resistant bacterial and viral strains, partly caused by drug overuse. Because the development of new drugs has not in all cases kept up with the emergence of new resistant strains of pathogens, such strains cause thousands of deaths annually, many in hospitals. Also, most synthetic drugs have more or less severe side effects, which affect a considerable number of patients. In spite of these drawbacks, the health benefits of antibiotics to humans and their domestic animals can hardly be overestimated. It has become apparent to many medical microbiologists and health professionals that besides synthetic drugs, which inhibit the replication of pathogenic microorganisms in a specific way, there may be a place for less specific antimicrobial compounds, microbicides, which kill the pathogens on contact. Microbicides could act in concert with specific antibiotics, launching a two-pronged attack on the invading pathogens. Direct killing, in addition to growth inhibition of pathogens, might make the formation of antibiotic-resistant strains less likely. Due to their broad antimicrobial actions, resistance to microbicides is rarely observed. The success of antibiotic drugs is due to the fact that our knowledge of their actions is based on a solid scientific ground. Their actions are in most cases predictable and their side effects known, because they have undergone a thorough scientific scrutiny, for both safety and activity, before being applied to the general public. In contrast, the use of natural health remedies was for a long time mainly based on anecdotal evidence and on accumulated experience of their beneficial effects obtained over centuries. The knowledge was mostly empirical. Recently, and mostly during the past few decades, the antimicrobial actions of the natural compounds, which originate in both the animal and the plant kingdom, have been studied by modern scientific methods similar to those applied in the study of synthetic drugs. This research has confirmed and extended the prior empirical knowledge of their antimicrobial activity.

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Introduction

In this book, scientific studies of the antimicrobial actions of two groups of naturally occurring organic materials are reviewed, namely lipids and essential oils. Lipids are diverse constituents of plants and animals which are insoluble in water but soluble in nonpolar organic solvents such as ethanol and ether. The main types of lipid are fats and oils, phospholipids, waxes and steroids. These lipids have various functions in the body. Animal fats and vegetable oils are triglycerides composed of fatty acids and glycerol and are a source of energy. Phospholipid molecules contain two fatty acids and are a major component of cell membranes. The hydrolytic products of triglycerides and phospholipids, particularly the fatty acids, have antimicrobial activities. In addition to being natural compounds they have the advantage of being both environmentally safe and generally harmless to the body in concentrations which kill pathogenic microbes. They are nonallergenic and are fully metabolized in the body. Lipids, particularly triglycerides, are abundant in nature and are an inexpensive source of antimicrobial products. The first part of this book deals with various aspects of lipids as antimicrobial agents, beginning with an examination of the chemical nature of lipids in Chapter 1. The history of lipids as antimicrobial agents, from the discovery of the antibacterial activity of natural soaps in the 1880s until 1960, is told in Chapter 2, followed by a discussion in Chapter 3 of more recent studies of the antibacterial, antiviral and antifungal actions of lipids. After chapters on antimicrobial lipids in mother’s milk (Chapter 4) and the skin (Chapter 5), Chapter 6 looks at the role of lipids in the natural host defence against pathogenic microorganisms, discussed in the context of other factors of the innate immune system. Recent studies strongly support earlier observations that natural fatty acids on the surface of the skin and mucous membranes contribute significantly to the host defence against infections by pathogenic microbes. Triglycerides in breast milk are hydrolysed by lipases in the gastrointestinal tract of infants, where they release free fatty acids, which seem to have an important protective effect against enteric pathogens. It has been suggested that the natural protective function of lipids could be enhanced by prophylactive or therapeutic application of drugs containing lipids as active ingredients. Chapter 7 reviews the application of lipids in pharmaceuticals, cosmetics and health foods and their possible use in therapeutics. A broad overview is given not only of antimicrobial activity but also of other health-related functions of lipids. Finally, Chapter 8 discusses antimicrobial lipids as disinfectants, antiseptics and sanitizers, for example in the food industry and in the home. The advent of antibiotics and inexpensive, synthetic detergents in the middle of the twentieth century caused a decline of interest in common hygiene outside of the hospital setting, for example in the home and in public places such as schools. The problem of drug-resistant pathogens and of environmental hazards caused by some synthetic disinfectants has led to an awareness of the advantage of using natural and environmentally friendly disinfectants and sanitizers. Thus, antibacterial lipids could, for example, be used to reduce the risk of contamination by foodborne pathogens in the kitchen and in food-preparing and food-processing facilities. Essential oils of flowering plants are secondary metabolites which are a part of the defence system of the plants, defending them against herbivorous animals and microorganisms. They have been used as health remedies for centuries but until recently little scientific research was carried out to establish the antimicrobial effect of essential oils and their chemical components. In the past few decades, a vast amount of scientific data has been collected on this subject and the second part of the book gives an overview of the current knowledge of the antimicrobial and biological functions of essential oils. Chapter 9 reviews

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Introduction

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the chemistry and bioactivity of essential oils and their use in food and cosmetic products. Chapter 10 describes the antiviral activity of essential oils and their effect in treatment of herpes simplex. Finally, Chapter 11 gives a comprehensive overview of the antibacterial and antifungal effects of essential oils, their use in pharmaceutical formulations and their clinical efficacy and toxicity in humans and animals. Although focussing on the antimicrobial action of lipids and essential oils, the book also describes various other health-related aspects of these natural products. It thus gives comprehensive and detailed information on the biological effects of lipids and essential oils based on the results of scientific research. Each chapter stands by itself and need not be read in the context of the others. Therefore, the chapters do not have to be read in sequence starting at the beginning of the book. There is thus a certain degree of overlap of data between chapters, but not redundancy, since the same data are viewed from different points of view and in different contexts by the different authors. Although written by scientists and aimed primarily at health professionals, the book is written in language which should be understandable to readers in general. It should be of interest to anyone concerned about health issues and particularly to those who are conscious of the benefits of health food and natural products. Halldor Thormar Reykjavik, June 2010

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1 Membranes as Targets of Antimicrobial Lipids Peter J. Quinn Department of Biochemistry, King’s College London, London, United Kingdom

1.1 1.2 1.3

1.4

1.5

1.6 1.7

Introduction Oil and Water Don’t Mix! Polar Lipids 1.3.1 The Amphiphilic Character of Polar Lipids 1.3.2 Hydrophobic Constituents of Lipids 1.3.3 Polar Groups of Complex Lipids Properties of Surfactants 1.4.1 Critical Micelle Concentration 1.4.2 Aggregation of Surface-Active Molecules 1.4.3 The Influence of Solvent Cell Membranes 1.5.1 Membrane Lipids 1.5.2 Lipid Domains in Membranes 1.5.3 Membrane Proteins 1.5.4 Membrane Stability 1.5.4.1 Membrane Lipid Phase Behaviour 1.5.4.2 Membrane Lipid Homeostasis in Homoiothermic Organisms 1.5.4.3 Membrane Lipid Adaption in Poikilothermic Organisms The Action of Antimicrobial Lipids on Cell Membranes Conclusions

Lipids and Essential Oils as Antimicrobial Agents
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1.1 Introduction Certain lipids are known to inhibit growth or kill microbes. A wide variety of lipids have been tested and they vary widely in chemical structure and efficacy. Because of the lack of a systematic relationship between lipid structure and antimicrobial activity an explanation of their mode of action is problematic. The two possible molecular mechanisms to account for the antimicrobial action of lipids are 1) a specific interaction with sites within the microorganism that influences biochemical functions and loss of viability or 2) a general nonspecific interaction that perturbs the structure of the microorganism, thereby inhibiting normal physiological functions. Lipids are a diverse class of compounds that defy definition by simple chemical characteristics but can be broadly categorized by their solubility in solvents of relatively low polarity. In this way they can be readily distinguished from the other constituents of living cells, such as nucleic acids, carbohydrates and proteins. Indeed, this is the operational basis for the extraction and purification of lipids from biological tissues. The conventional methods of lipid extraction employ solvent mixtures with a relatively polar solvent, initially to loosen up the tissue and dislodge lipids from their interaction with other cellular constituents and culminate in isolation of the lipids in a two-phase system in which the polar molecules partition into an aqueous phase [1, 2]. Within the general class of lipids, subdivisions are recognized on the basis of their relative solubility in solvents of low polarity, or to put it another way, solubility in solvents of different polarities. Again, no systematic chemical criteria can be adopted to account for solubility of lipids in solvents of different polarities. However, polarity of solvents can be defined by objective criteria [3, 4] and solubility of lipids in solvents can be measured by a variety of biophysical parameters. This chapter will provide an account of lipid solubility in solvents of different polarities and explain the general chemical principles that govern this property. The relevance of lipid solubility to antimicrobial action will be discussed in the context of the role of lipids in the structure of cell membranes of living organisms and how these structures are disrupted by antimicrobial lipids.

1.2 Oil and Water Don’t Mix! We are all familiar with the old adage that oil does not mix with water. Equally, we know intuitively that if a drop of ink is placed in a beaker of water the ink will diffuse out from the concentrated drop, eventually distributing randomly throughout the aqueous phase. Both of these situations have expressions in the Law of Thermodynamics, which states that all systems move to their state of lowest free energy, and in the cases we are considering, to a more random and chaotic state. The formulation is:
G =
H − T
S

(1.1)

where
G is the change in free energy of the system,
H is the change in heat, T is the absolute temperature and
S is the change in entropy. The negative sign on the T
S component signifies a spontaneous change from a more ordered to a disordered state.

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At first sight there seems to be a contradiction in the two examples given above. The twophase system of oil and water appears to be a perfectly ordered system, yet clearly this is the equilibrium position. To understand why this is the lowest free energy of the system we need to consider the consequences on the order of the molecules if we attempt to place oil into an aqueous environment. Oils are largely composed of hydrocarbon, and hydrocarbons are nonpolar, since they have electron distributions about the constituent atoms that are relatively even. By contrast, water is highly polar, with electrons attracted to the oxygen atom generating a surfeit of negative charge (a ␦-negative charge) and creating a deficiency of negative charges (␦-positive charges) associated with the two hydrogen atoms. This is the basis of molecular polarity. When hydrocarbon is exposed to water the molecules of water in contact with the hydrocarbon lose their freedom to interact with like polar water molecules and consequently become ordered. It is this molecular order that results in a decrease in entropy and a consequent increase in free energy of the system. Lipids of biological origin are not composed purely of hydrocarbon, and constituent atoms like oxygen and nitrogen bring about an asymmetric distribution of electrons within the lipid molecule. This provides opportunities for polar interactions with water, thereby increasing the entropy of the system. We next consider the origin of polarity of biological lipids and the common chemical strategies used in nature to achieve a polar character.

1.3 Polar Lipids Living cells do not synthesize pure hydrocarbons directly, although compounds such as methane and ethane are byproducts of lipid metabolism of some microorganisms. In general, therefore, the lipids of living cells contain electrophilic atoms like oxygen, nitrogen, sulfur, phosphate and so on that confer a polar character on the lipid. The presence of polar groups on lipids renders them less soluble in nonpolar solvents and induces them to assemble into characteristic aggregates or dispersions in water rather than forming a two-phase oil and water system. 1.3.1

The Amphiphilic Character of Polar Lipids

The presence of a polar group in a lipid has considerable influence on the properties of the molecule apart from its solubility in water. This is particularly the case with complex lipid molecules in which domains of nonpolar hydrocarbon within the molecule are separate from polar moieties. This arrangement confers on the lipid the physical properties of an amphiphile (Greek: amphi, on both sides; philos, loving), in which the hydrocarbon portion achieves a lowest free energy in a nonpolar environment and the polar group can be hydrated. Amongst the weakest amphiphiles in biological tissues are the fats and oils, which largely make up the nutritional reserves of triacylglycerides. Such lipids generally form two-phase systems in water because the carbonyl oxygens of the acyl ester bonds linking the fatty acids to the glycerol backbone are not sufficiently polar to balance the remaining hydrocarbon character of the lipid. This is reflected in the locations of oils and fats within cells in phase-separated droplets bounded by a membrane and sequestered from contact with water.

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Lipids and Essential Oils as Antimicrobial Agents

As the balance of hydrophilic affinity created by the hydrocarbon component increases, so does its tendency to interact with water. Polar lipid molecules are therefore able to bridge a hydrophilic and a hydrophobic environment and are said to be surface-active or posses the properties of a surfactant. 1.3.2

Hydrophobic Constituents of Lipids

The biosynthesis of lipids is conducted primarily by two metabolic pathways: the pathway for synthesis of sterols and related prenyl compounds, and fatty acid synthase. The products of both pathways provide lipids that are common constituents of cell membranes. Sterols are all derived from isoprene substrate and perform essential structural and signalling roles in eukaryotes. In vertebrates cholesterol is the major structural sterol, whereas in plants a variety of sterols are found, including stigmasterol, sitosterol and camposterol, with other minor sterols. The sterol found mainly in microorganisms is ergosterol. Sterols are weakly polar, with an amphiphilic balance favouring hydrophobic interactions. The dominant feature of their structure is the polycyclic ring and the associated hydrocarbon side chain. The polar affinity resides in a hydroxyl group located on the sterol ring. Other important prenyl-derived compounds are the ubiquinols and plastoquinols, which are typified by relatively long hydrocarbon chains attached to a fully substituted benzoquinone ring system conferring a weakly polar character to the lipid. The products of fatty acid synthase are fatty acids with a hydrocarbon carbon chain length of 16 carbons – palmitic acid. Palmitic acid can be elongated by the sequential addition of two-carbon units up to aliphatic carbon chain lengths of C24. Oxidative processes are also able to desaturate fatty acids at specific locations in the hydrocarbon chain, resulting in the insertion of cis-double bonds. The first double bond is inserted in the centre of the palmitate chain at position ␻9 (the nomenclature counts from the terminal methyl carbon because biochemically this represents the sequence of desaturation of the alkyl chain) and subsequent desaturation occurs at ␻6 and then ␻3. Only plants are able to perform the last two desaturations, so that linoleic and linolenic acids are essential fatty acids for animals and represent vitamin F [5]. Other modifications of the aliphatic chain include the formation of branched chains by the attachment of methyl side-chain groups. Oxidative reactions of arachidonic acid represent the precursors of important signalling compounds, comprising the prostaglandins, thromboxanes and leucotrienes. Fatty acids represent basic building blocks of complex polar lipids that are major constituents of biological membranes. The two principle classes of complex lipid that incorporate fatty acids are the glycerolipids and sphingolipids. The backbone of the glycerolipids is the anomeric polyol glycerol. Long-chain fatty acids are esterified to the C1 and C2 positions of the glycerol to form a diglyceride. Fatty acids are attached to the long-chain base, sphingosine, by an N-acyl linkage to form the basic building block of the sphingolipids, ceramide. The chemical structures of these hydrophobic components of complex polar lipids are illustrated in Figure 1.1. Another group of long-chain hydrocarbons that are widely found in both eukaryotes and prokaryotes are waxes. These relatively nonpolar lipids constitute components of the so-called neutral lipid fraction and are composed of long-chain n-alkanolic acids and nalken-1-ols of an even number of carbon atoms ranging in length from C12 to C32. Carbon chains of between C20 and C24 are commonly found in the acyl portion, whilst C24 and

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Diglyceride

O R

O O H O

Ceramide H OH O NH H

R

O Cholesterol H

H H

H

HO H

Figure 1.1

The chemical structures of the hydrophobic components of complex polar lipids.

C28 predominate in the alcohol component. A certain amount of unesterified hydrocarbon material may also be associated with wax esters as well as fatty aldehydes. Waxes can fulfil a protective role and the hydrophobic character of their structure can act as a reservoir that traps lipids with potential antimicrobial activities. Other lipids classified with the neutral lipids are carotenoids, sterol esters and glycerides. 1.3.3

Polar Groups of Complex Lipids

Polar groups that are responsible for the amphiphilic properties of complex cellular lipids are generally attached to hydroxyl groups of the diacylglycerol or ceramide. The type of polar group is used to designate the class of complex lipid. The structures of polar groups that represent the basis of classification of complex lipids are shown in Figure 1.2. Arguably the most abundant polar lipids in nature are the glycosylglycerides, in which the diacyl glycerol is acylated to hexose sugars. While many different types of hexose may be attached glysidically to the C3 position of the glycerol, galactose is the most common in plant and algal systems. Mannose and glucose are more frequently encountered in bacterial species. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols are the major lipids of the chloroplast thylakaoid membranes and their polar character is due to hydration of the sugar groups. The extent of hydration is increased in another lipid, 6sulphoquinovosydiacylglycerol, by the presence of a sulphonate group on the sugar, and this lipid has been identified as a constituent of the membranes of all photosynthetic plants, algae and bacteria so far examined. The most ubiquitous complex polar lipids are the phospholipids. The basic structure is phosphatidic acid, in which a phosphate group is esterified to the C3 of the diacylglycerol. Although this phospholipid is present only in minor proportions in tissue lipid extracts it is

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Lipids and Essential Oils as Antimicrobial Agents O R1

O

Phosphatidylcholine

O P O O– O H

O R2

N+

R1

R2

O O R1

O O

R2

R1

O R2

Phosphatidylserine O O– H

O P O O– O H

R1

R2

OH HO O

O R2

OH OH

OH OH

OH

+

NH4 O– O S O

O HO O

O R2

OH O

OH

O

R1

OH HO O

O

O H

NH3+

O HO O P O O– O H Na+

O H

O

OH

O OH Sulfoquinovosylidiacylglycerol

O Phosphatidylglycerol

O O

R1

Phosphatidylinositol

O O

Digalactosyldiacylglycerol O

NH3+

O O

OH

O O P O O– O H

R2

OH O

O H

Phosphatidylethanolamine

O O

HO O

O

O O R1

Monogalactosyldiacylglycerol

O

OH

O P O O– + O H Na

OH

O

Figure 1.2

The structures of polar groups of membrane lipids.

a pivotal intermediate in the biosynthesis of the major phospholipid classes. Thus, different groups attached to the phosphatidic acid define the particular class of phospholipid. The major classes of phospholipids are based on choline, ethanolamine, serine, glycerol and inositol substituents on the phosphate group of phosphatidic acid. The amphiphilic property of the phospholipids is due not only to hydration of the negatively-charged phosphate but to hydroxyls, carboxyls and amino groups of the polar moieties. A major class of sphingolipid is based on attachment of a phosphocholine group to ceramide to form sphingomyelin. The remaining sphingolipid classes, however, are not phospholipids but rely on the sugar residues attached to the hydroxyl residue of ceramide for their amphiphilic properties. The attachment of a single hexose such as glucose or galactose to the ceramide backbone constitutes the class of cerebrosides. More complex glycosphingolipids are the gangliosides and globosides. These polar lipids contain branched-chain oligosaccharides with as many as seven neutral and amino sugars attached to the ceramide. The presence of such large polar groups renders these classes considerably less soluble in nonpolar solvents. In addition to the polar lipids normally found associated with living cells, a range of surfactant compounds are known to be biosynthesized by various microorganisms. Such biosurfactants have unique properties such as biodegradability and production under relatively benign environmental conditions and can be produced from substrates consisting of vegetable and even industrial waste materials. An important example of biosurfactants is the mannosylerythritol lipids produced by the yeast strain of the genus Pseudozyma [6, 7].

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The amphiphilic properties of these glycolipid biosurfactants are due to sugar residues, which are often coupled to acetate groups and acylated with one or more hydrocarbon chains of varying length. Amongst the features of useful detergent-like properties [8,9], the glycolipid biosurfactants have been shown to promote differentiation [10] and apoptosis [11] of immortalized tumour cell lines in tissue culture as well as to bind antibodies [12,13] and lectins [14].

1.4

Properties of Surfactants

As described in the preceding sections, the dominant feature of structural lipids found in living cells is that they are amphiphilic. However, the balance of hydrophobic and hydrophilic affinities within the lipids varies greatly depending on the extent of the hydrocarbon domain of the molecule and the affinity of the polar group for water. This balance influences the ability of the polar lipids to act as surfactants bridging the interface between hydrophobic and hydrophilic environments. 1.4.1

Critical Micelle Concentration

A useful parameter to compare the relative surface activities of surfactants is the critical micelle concentration. The parameter is defined as the concentration of polar lipid in free solution that is in equilibrium with aggregates of surfactant. Where the solvent is water, the concentration of polar lipid in free solution decreases as the hydrophobic affinity within the molecule relative to the polar affinity increases. Surfactants will tend to concentrate at interfacial regions between water and nonpolar solvents or air as this will represent the lowest free energy of the system. There are two aspects to this reduction in free energy. The first has been described above and results from the removal of hydrocarbon from contact with water. The second is to lower the free energy of the interface between the two media; that is, a reduction in surface tension. When all the interface is occupied by surfactant molecules a further increase in surfactant concentration will result in the formation of aggregates of the surfactant as again the association of surfactant molecules is configured so that hydrocarbon is sequestered away from water and the polar groups of the surfactant are exposed on the outside so as to form a stable aggregate. A convenient method for measuring critical micelle concentration of a surfactant is to monitor the surface tension of water at an air interface with increasing concentration of surfactant in the subphase. This is illustrated in Figure 1.3. A comparison of the surfactant activity of common surfactants with the polar lipids of biological membranes shows that membrane lipids are only weakly surface-active. The critical micelle concentration for most membrane lipids is in the range of nM concentration. This means that membrane lipids are overwhelmingly present in aqueous systems in the form of aggregates. Biosurfactants, on the other hand, have considerably higher critical micelle concentrations. Mannosylerythritol lipids have critical micelle concentrations in the ␮M concentration range at interfacial surface tensions of about 30 mN/m [15]. This compares with the critical micelle concentration of typical domestic washing-up liquids, which are in the mM concentration range.

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Lipids and Essential Oils as Antimicrobial Agents

60 Surface tension, mN/m

c01

55

50

45

Critical micelle concentration

0.2

0.4

0.6 0.8 1.0 Ubiquinone-10,µM

1.2

1.4

Figure 1.3 Measurement of the critical micelle concentration of a surfactant by relating the decrease of the surface tension of formamide at an air interface as a function of surfactant concentration of the subphase. (Reprinted from [21]. With kind permission from Springer Science+Business Media.)

1.4.2

Aggregation of Surface-Active Molecules

Amphiphiles self-assemble in solvents into a variety of aggregates with normal or reversed curvature. The structures have been classified according to their morphology and range from dispersed micellar to cubic to hexagonal to lamellar phases [16]. The rich polymorphism displayed by surfactants arises from the wide differences in amphiphilic balance within the molecules. The polymorphism of these structures can be modulated by changing the polar interaction with the solvent or by altering the van der Waals interactions between the hydrocarbon residues of the surfactant. Thus surfactants exhibit lyotropic mesomorphism in response to the changing hydration of the polar head group. Likewise, thermotropic mesomorphism can be demonstrated as a consequence of changes in packing order of the hydrocarbon domain at different temperatures. This is illustrated by the simple binary mixture of glycerol monooleate. The temperature–composition phase diagram in the temperature range 20 to 105 ◦ C and water content 0 to 100% (w/w) was first published 25 years ago [17]. The phase diagram was subsequently extended to temperatures below 20 ◦ C, where complicated behaviour involving metastable phases was reported [18]. Nonionic detergents are known to have different effects on the phase behaviour of monoolein. For example, at full hydration at 20 ◦ C alkyl glucosides in mole fractions up to 0.25 in monoolein form a cubic phase of space group Pn3m determined from X-ray scattering methods [19]. In the presence of higher mole fractions of detergent a liquid–crystal bilayer is the preferred phase, with an intermediate cubic phase of space group Ia3d observed in mole fractions of 0.4 nonionic detergents. Somewhat different effects on the phase behaviour of monoolein/water systems have been reported for terpinen-4-ol, the active surfactant of tea-tree oil, as evidenced from optical and NMR studies [20]. The influence of the presence of 5 wt% terpinen-4-ol on the lyotropic phase behaviour of monoolein is shown in Figure 1.4. It can be seen that in low water contents (<5 wt%, D2 O) a micelle phase is formed by the monoolein, and this is not greatly altered by the presence of terpinen-4-ol. With increasing water content the

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Monoolein/D2 O

cubic

bilayer

micelle Monoolein/D2 O/terpinen-4-ol

cubic

bilayer

micelle

10

hexagonal-II

20

30

hexagonal-II + D2 O 40

% D2O

Figure 1.4 The influence of the presence of 5 wt% terpinen-4-ol on the lyotropic phase behaviour of monoolein. (Reprinted with permission from [20]. Copyright 2002 American Chemical Society.)

lamellar liquid–crystal phase is stabilized up to about 10 wt% water before forming the cubic phase. The major effect of the terpinen-4-ol is to destabilize the cubic phases and induce a hexagonal phase of type I. This effect is due to the partitioning of the terpinen4-ol into the aqueous interfacial domain, where it serves to expand the area of lipid–water interface. The general conclusion from studies of surfactants in aqueous systems is that the structure formed by the surfactant in concentrations above the critical micelle concentration depends primarily on the amphiphilic balance within the molecule. This can be illustrated in the case of a common membrane lipid, phosphatidylcholine. This phospholipid forms only lamellar phases in water over a wide range of temperatures. Removal of one of the fatty acyl residues from the glycerol produces lysophosphatidycholine. This nomenclature is in deference to the fact that the lipid will cause haemolysis of erythrocytes by a detergent action on the cell membrane. The dramatic shift in amphiphilic balance resulting from removal of one fatty acyl residue converts a weak surfactant into a relatively strong detergent. This is reflected in the type of aggregate formed by lysophosphatidycholine in water: hexagonal-I, which consists of tubes of lipid with acyl chains oriented into the interior and the alignment of the tubes in a hexagonal packing array. 1.4.3

The Influence of Solvent

A component of amphiphilic balance of surfactant molecules is the interaction of their polar groups with the solvent. The effect of the polarity of the solvent can be illustrated by the solubility of ubiquinone-10 in ethanol–water mixtures [21]. Figure 1.5 shows the concentration of ubiquinone-10 in the supernatant of centrifuged samples, judged by absorption at ␭ = 275, as a function of the volume of water added to an ethanol solution of the lipid. There is a marked decrease in solubility of ubiquinone-10 when the proportion of water in the solvent mixture exceeds about 12% by volume. Examination of the aggregated lipid by wide-angle X-ray scattering methods indicates that the benzoquinone ring of the molecule is not solvated in the aggregated form. Nevertheless, the demonstration shows that

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Lipids and Essential Oils as Antimicrobial Agents 2.0

1.5 Absorbance, λ275

c01

1.0

0.5

5 10 15 Volume% water in ethanol

20

Figure 1.5 The effect of polarity of the solvent on the solubility of ubiquinone-10 in ethanol–water mixtures. (Reprinted from [21]. With kind permission from Springer Science+Business Media.)

increasing solvent polarity causes aggregation of the weak surfactant as the hydrocarbon component is forced to exclude increasingly polar solvent. Modulation of the electrostatic charge of ionic surfactants greatly influences the phase behaviour of amphiphiles. The phospholipids of cell membranes contain charged phosphate groups and some classes contain additional amino or carboxyl groups. Counter-ions in the aqueous medium are known to play an important role in the phase behaviour of the lipids in the membrane bilayer matrix. An example of this is the physiological process of domain structure and fusion between bilayer membranes, which involves the creation of a nonlamellar transition state within the phospholipid bilayer. Calcium is a potent membrane fusogen which, by binding to the phosphate groups, reduces charge repulsion between adjacent bilayers, and by promoting aggregation is able to bridge the bilayers and promote fusion through the creation of defects in the lamellar structures [22]. A component of the charge neutralization of the phospholipid polar group by interaction with calcium is an alteration of the structure of the water layer that hydrates the interface. The thermodynamics of lipid hydration has been considered in thermal studies of multibilayer liposomes of membrane polar lipids that each take up between 10 and 30 molecules of water depending on the nature of the polar group and the state of the alkyl chains [23]. Measurements of the partial molar free energies and enthalpies of these swelling systems indicate that the first four water molecules hydrate the lipids in a favourable enthalpic interaction but the partial free energies and enthalpies of additional water molecules have opposite signs. There is also evidence that additional hydration involves changes in the thermal excitation of the lipid degrees of freedom.

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1.5

11

Cell Membranes

As indicated in Section 1.3, there is a wide range of weakly polar lipids associated with cell membranes of living organisms. The other major component of membranes is the protein; because most incorporate sugar residues attached to the polypeptide chain, these are mainly glycoproteins. Since cell membranes represent important targets for antimicrobial lipids, we next consider the structure of cell membranes and the factors that govern their stability. The contemporary view of the structure of biological membranes is formalized in the fluid-mosaic model proposed nearly 40 years ago [24]. The polar lipids are said to be arranged in a fluid bilayer structure which acts as a matrix for the orientation and organization of the different intrinsic and extrinsic proteins. While this view has proved to be reasonably durable, much greater detail has emerged about the disposition of the components on either side of the bilayer and within lateral domains of the bilayer. Furthermore, the notion that the lipid bilayer matrix is a fluid structure has been modified by the realization that various degrees of order in the lipids are created by the presence of sterols when they interact with sphingolipids [25]. 1.5.1

Membrane Lipids

The types of polar lipid found in biological membranes were seen in Section 1.3 to include phospholipids, glycolipids and sterols. Apart from sterols and other neutral lipids, each of the major classes of polar lipid consists of a family of molecular species defined by the type of hydrocarbon chain associated with the lipid backbone. In the case of the diacylglycerophospholipids, the fatty acids acylated at the sn-1 and sn-2 position of the glycerol differ in length, degree of cis-unsaturation and position of attachment to the glycerol. In some members of the family the fatty acids may be branched-chain or they may be attached by ether or vinyl ether rather than ester bonds to the glycerol. The importance of this complexity can be understood by the differences that the hydrocarbon component of the lipid confers on the physical properties of the lipid. One of the most important features governing the morphological characteristics of phospholipid assemblies in aqueous systems is the presence of cis-unsaturated bonds. These bonds, as opposed to double bonds in the trans-configuration, introduce a kink into the chain that prevents the close parallel alignment required to maximize van der Waals cohesive bonds between them. As a consequence, the temperature at which the chains are transformed from a solid to a liquid is greatly reduced. In the context of a lipid bilayer the transition between ordered and liquid–crystal lamellar phases as well as between lamellar and nonlamellar phases is determined by the number and location of cis-unsaturated bonds in the fatty acid residues associated with the phospholipid. Characteristics of the phases adopted by membrane lipids in aqueous systems are illustrated in Figure 1.6. A review of the behaviour of membrane lipids and their arrangement in the structure has been published recently [26]. With the advent of sophisticated mass-spectrometric methods for analysis of the lipid composition of biological membranes [27] it has been recognized that each morphologically distinct membrane contains a complex and distinctive lipid composition. In many membranes, hundreds of molecular species of lipids have been characterized. Moreover, the constituent lipids of each membrane appear to be preserved within relatively narrow

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Lipids and Essential Oils as Antimicrobial Agents Hexagonal-I

Lamellar

Hexagonal-II

Bicontinuous cubic phase

Figure 1.6

Characteristics of the phases adopted by membrane lipids in aqueous systems.

limits by biochemical mechanisms that are, as yet, not completely understood. The processes of membrane lipid turnover and homeostasis raise questions as to the reasons for the diversity of membrane lipid compositions. Why, for example, do the cells of higher organisms maintain such a complex lipid matrix despite being protected from variations in their environment by the homeostatic regulation of surrounding extracellular fluids or serum and the composition of the cytosol? We do not know whether the maintenance of narrow ranges (homeostatic regulation) of any particular molecular species of lipid in the membrane is critical to the cell or the functions performed by the constituent membranes and how much redundancy is incorporated into the system. There are also uncertainties as to whether lipid complexity is predicated by the assortment of proteins in particular membranes or whether lipids have their own rules for assembling which govern, in turn, the way proteins are ‘passively’ inserted into the matrix. Lipid homeostasis for a complex mixture represents a cost in terms of metabolic expense and gene diversity, which encodes all enzymes responsible for catalysing the biosynthetic and degradation pathways. Multiple cross-regulation would be expected to achieve the characteristic composition of membrane. The maintenance of a complex lipid composition requires regulated pathways to repair the alterations (literally ‘homeostasis’) induced by the ‘activated enzymes’ perturbing the cell membranes. Methods of establishing the physical consequences of the diversity of lipid composition have not kept pace with the biochemical characterization of this diversity in particular membranes. The main reason for this is that methods employed to characterize the conformation and structure of the membrane lipid matrix are averaging techniques, such as spectroscopy, that are unable to distinguish subtle differences in local environment created by domains dependent on lipid complexity. As a result, much information is indirect

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and based on the construction and examination of models to simulate the lipid matrix of biological membranes. Frequently such models diverge considerably from the real world because of the low energy barriers that separate conformational states in the complex mixtures which represent biological membranes that are less likely to occur in defined lipid mixtures. Furthermore, in terms of representation, some molecular species may be present in relatively minor proportions and would not be expected to greatly influence the phase structure of the membrane lipid matrix. Others, such as cholesterol and sphingomyelin in the plasma membrane, may dominate the lipid composition of the membrane and exert a major impact on the structure and properties of the membrane. Clearly, characterizing the physical consequences and influences of individual molecular species of membrane lipid on the structure and properties of the bilayer matrix remains a considerable challenge. 1.5.2

Lipid Domains in Membranes

The segregation of the lipids of cell membranes into separate domains is now known to underlie membrane functions like signal transduction, fusion and so on. [28, 29]. This realization has come about through the characterization of so-called liquid-ordered phases formed between choline phosphatides and cholesterol. Such phases are created by specific interactions between the molecules which segregate from domains of fluid lipids to form a platform or raft into which lipid-anchored membrane proteins are partitioned. The segregation of these proteins from the fluid phase of the membrane appears to be required for them to perform their function. Cell membranes can be fractionated according to their solubility in mild detergents [30]. The detergent-insoluble fraction floats at low density upon gradient centrifugation. The membranes can easily be harvested, the residual detergent removed and the resulting membrane further purified or subfractionated. The membrane fractions isolated in this way are referred to as rafts and take the form of vesicles about 200 nm in diameter [31]. Membrane lipids act as platforms for the assembly of receptors on one side of the membrane and appropriate effecter proteins on the opposite side. This arrangement allows signals generated when a ligand binds to its receptor to be transduced across the membrane to the biochemical apparatus responsible for setting the physiological response in train. The polar lipids of the raft serve as filter devices, in order to include particular membrane proteins and exclude others. The lipids of the raft membrane are distinctive from those of the surrounding membrane and a specific interaction between the lipids is believed to be the mechanism underlying their segregation. The characteristic feature of the lipid composition of membrane rafts is the predominance of phospholipids with saturated hydrocarbon chains and the high proportion of sterol. This is particularly evident in the molecular species of sphingomyelin in membrane rafts isolated from erythrocyte ghost membranes [32]. To investigate the factors that govern the partition of sphingomyelin into the rafts the molecular species of sphingomyelin recovered in the detergent-resistant membrane fractions were compared with those in the membrane ghosts from which they were derived. The fatty acids in amide ester linkage to the sphingosine isolated from human erythrocyte ghost membranes and corresponding raft fractions are shown in Table 1.1. Saturated molecular species of sphingomyelin dominate the raft membrane fraction at the expense of monoenoic fatty acids. This is achieved by an approximately threefold enrichment of C22 : 0 and C24 : 0 molecular species of sphingomyelin and the

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Table 1.1 N-acyl-linked fatty acids of sphingomyelin in human erythrocyte ghosts and detergent-resistant membranes isolated from them by treatment with Triton X-100 and fractionation on a density gradient. Human erythrocyte sphingomyelin N-acyl fatty acid

Ghost membrane

Raft membrane

C16 : 0 C18 : 0 C18 : 1 C18 : 2 C20 : 0 C22 : 0 C24 : 0 C24 : 1 C26 : 0 C26 : 1 Total

41.45 ± 5.68 6.25 ± 0.74 1.15 ± 0.21 0.10 ± 0.01 0.70 ± 0.01 5.78 ± 0.48 20.02 ± 2.35 24.02 ± 1.87 0.33 ± 0.04 0.19 ± 0.01 100

8.63 ± 0.74 6.36 ± 0.66 1.49 ± 0.21 0.18 ± 0.13 2.22 ± 0.03 14.72 ± 2.02 62.10 ± 5.68 1.47 ± 0.19 2.78 ± 0.21 0.05 ± 0.01 100

Saturated Monounsaturated Polyunsaturated

74.54 25.36 0.10

96.81 3.01 0.18

Data from [32].

almost complete exclusion of molecular species of sphingomyelin associated with C24 : 1 fatty acid. The raft fractions isolated under mild conditions appear to represent individual domains within the membrane. Evidence for this is that detergent-resistant membranes retain their original asymmetry and can be subfractionated by immunoprecipitation methods into vesicles that contain unique sets of antigens. In the case of neuronal cells, separation of vesicles with prion protein from vesicles displaying Thy-1 can be achieved. A similar segregation of these antigens is observed in the intact cell membrane [30]. Lipid analysis of prion protein and Thy-1 vesicles show that the two raft membranes have different lipid compositions and in turn these are different from that of the detergent-resistant membrane fraction from which they were derived [33]. Thus prion protein vesicles contain significantly more unsaturated, longer-chain lipids than Thy-1 vesicles and have a fivefold greater content of hexosylceramide. These results lead to the conclusion that unsaturation and glycosylation of lipids are major sources of diversity of raft structure. While sphingomyelin and cholesterol have tended to achieve prominence in raft lipid composition, more recent studies have indicated that glycerophospholipids and diacylglycerols are also constituents of raft fractions. One method that has been used to identify such components is the detection of lipids from radioactive precursors that are isolated in raft fractions [34]. The method involves feeding cells with radiolabelled glycerol, fatty acids or water-soluble polar groups and identifying the complex lipid into which they are biosynthetically incorporated. Using this approach it was shown that raft fractions derived from human leukaemic T-cell line Jurkat had a considerably higher cholesterol content than

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the parent membrane and that polar lipids incorporating [3 H]-glycerol were present. These glycerophospholipids included choline, ethanolamine, serine and inositol phosphoglycerides, with a preponderance of phosphatidylcholine and phosphatidylserine. Incorporation of radiolabelled fatty acid precursors into the phospholipids showed preferential labelling of raft lipids with saturated fatty acids such as palmitic and stearic acids, rather than oleic, linoleic and arachidonic acids. These results are consistent with other studies indicating that the lipids isolated in the raft fractions contain predominantly saturated molecular species of membrane lipids [35, 36]. 1.5.3

Membrane Proteins

Proteins are major constituents of membranes and vary in proportion to the polar lipids from 0.25 wt/wt lipid in central-nerve myelin to more than 3.5 wt/wt lipid in the inner mitochondrial and photosynthetic thylakaoid membranes. The association of the protein with the lipid bilayer matrix can occur via polar interactions, in which case the protein can be dislodged from the structure by modulating the ionic environment. These proteins are referred to as extrinsic or peripheral membrane proteins. Another group of proteins are the intrinsic proteins and these are in contact with the hydrocarbon chains of the lipids. They require detergents to extract them from the lipid bilayer matrix. A third group are lipid-anchored proteins, which are essentially water-soluble proteins that incorporate a covalently-bound lipid which anchors them to the bilayer. A typical anchor tethering such proteins to the external surface of the plasma membrane is glycerylphosphorylinositol, forming the (so-called) GPI-anchored proteins [37]. Lipid anchors associated with the proteins on the cytoplasmic surface of membranes are saturated fatty acids and isoprenyl groups [38]. Because there are no proteins or groups of proteins common to biological membranes it is agreed that proteins are not required to organize or direct the structure of membranes. This means that the structure of membranes is not a process that is directly under genetic control and the assembly of components is driven by entropic forces. Nevertheless, membrane proteins can exert an essential role in the differentiation of particular membrane structures. Examples of such structures include intercellular junctions and the grana stacks of chloroplast thylakoid membrane. Indeed, such structures serve to confine different proteins to domains within the same contiguous membrane. The orientation of proteins within the bilayer matrix is vectoral. The unique arrangement of each protein with respect to the lipid bilayer is necessary to perform transport and other biochemical functions in a unidirectional manner. The structure of membranes is established during membrane biogenesis, which occurs by a process of expansion and differentiation of preexisting membrane. This involves incorporation of newly synthesized proteins and lipids, the post-translational modification of the proteins and metabolic retailoring of the lipids. 1.5.4

Membrane Stability

The thickness of biological membranes is in the order of 5 to 6 nm, while the surface area can be very large, considering for example the size of an ostrich egg. Soap bubbles are another example of stable thin structures held together by the forces holding the weak

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surfactants of membrane lipids together. These thickness-to-area-ratio arguments suggest a structure that is quite robust, but the cohesive forces keeping the molecules together are in fact relatively weak van der Waals cohesive forces. These forces arise from fluctuating dipoles created by the asymmetric electrostatic charge distribution about all atoms and the interacting force decreases according to the sixth power of the distance separating the atoms. Although they are relatively weak forces they add up over the surface of adjacent hydrocarbons to provide strong cohesion between lipid molecules. This, together with the entropic forces associated with exclusion of water considered in Section 1.2, is why biological membranes are inherently stable structures. This stability may seem to contradict the dynamic properties of biological membranes. Many cellular processes, including membrane biogenesis, secretion, endocytosis and so on, require fusion between membranes. In order for membrane fusion to take place in an orderly manner, specific membrane proteins are involved to make sure that indiscriminate fusion is avoided. Secondly, the bilayer arrangement at the point of fusion must be disturbed and transient nonbilayer arrangements of the lipids are required to complete the fusion process. The question is, how can an essentially stable structure have a built-in mechanism to cater for a dynamic process like fusion? The answer is found in the diverse amphiphilic balance of the lipids that constitute the membrane bilayer matrix. It has long been known that the total polar lipid extracts of biological membranes do not form bilayer structures when dispersed in aqueous media under physiological conditions [39]. Instead they form a range of structures from bilayer to hexagonal-II and cubic arrangements. We can conclude from this observation that a proportion of the lipids found in cell membranes are inherently unstable in a bilayer configuration under physiological conditions and that interaction with other membrane components is required for them to conform to a bilayer arrangement. The membrane lipids that, when dispersed in pure form in aqueous media, assume a nonbilayer structure (mainly hexagonal-II structure) are the phosphatidylethanolamines and monohexosyldiacylglycerols. Indeed, all biological membranes contain a proportion of membrane lipids that form nonbilayer structures. It can now be seen that dynamic processes like the fusion between membranes can be accomplished by controlled dissociation of nonbilayer-forming lipids from the constraints that normally preserve them in a bilayer configuration. Most biological membranes have a much greater proportion of nonbilayer-forming lipids than would be required to participate in membrane fusion events, so other functions may be ascribed to these lipids. One such function is to seal the interface between intrinsic membrane proteins and the lipid bilayer matrix. Because the amphiphilic balance in nonbilayerforming lipids favours a hydrophobic affinity they are ideally placed to occupy the voids created by the irregular polypeptide surface, thereby blocking the passage of water-soluble solutes across the membrane. Their function could be seen as sealing the intrinsic proteins into the lipid bilayer matrix. To put this in context, the inner mitochondrial and chloroplast thylakoid membranes both have a relatively high proportion of nonbilayer-forming lipids: cardiolipin in the case of mitochondrial membrane and monogalactosyldiacylglycerol in thylakoid membrane. These membranes have a high intrinsic membrane protein-to-lipid ratio, are required to be impermeable to protons and, despite coming into close proximity with adjacent membrane surfaces, do not fuse. The consensus picture that emerges from consideration of these factors is that biological membranes are stable barriers to the passive diffusion of water-soluble solutes and yet are

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potentially unstable due to possession of nonbilayer-forming polar lipids that, if separated into pure phases, could destroy the properties of the membrane and ultimately lead to loss of viability of the cell. The critical factor that preserves the functional integrity of cell membranes is the maintenance of a balance of components within the structure that prevents uncontrolled dissociation of nonbilayer-forming lipids from their interaction with molecules constraining them into a bilayer configuration. Because environmental factors play a critical role in the tendency of nonbilayer-forming lipids to form nonbilayer structures there is a dichotomy between homoiothermic and poikilothermic organisms in how they handle this process. 1.5.4.1

Membrane Lipid Phase Behaviour

The phase that membrane lipids form when dispersed in physiological media at the growth temperature depends on both the type of hydrocarbon component and the hydration and charge of the polar group. In general, the molecular species of choline-, serine-, glyceroland dihexosyl-based polar lipids extracted from biological membranes form fluid bilayer structures. The molecular species of ethanolamine- and monohexosyl-based polar lipids and cardiolipins form hexagonal-II structures. The factors that determine the particular phase that will form include the packing constraints determined by the structure of the lipid, and environmental factors like temperature, pressure, water activity and solutes [40]. Nonbilayer-forming lipids will form bilayer structures at temperatures below the growth temperature. The temperature at which these lipids undergo a bilayer to nonbilayer transition is increased by increasing the length of the hydrocarbon chains, decreasing the number of cis-unsaturated bonds and increasing the hydration of the head group [41, 42]. These thermotropic and lyotropic effects have considerable influence on the stability of biological membranes when shifted from environmental conditions that support growth of cells. Consider the consequences of exposing cell membranes to low temperatures [43]. The lamellar gel to liquid–crystal phase transition temperature of nonbilayer-forming lipids is considerably higher than the same transition observed in bilayer-forming lipids with equivalent hydrocarbon-chain components. This means that in mixtures of molecular species of lipid that occur in cell membranes, cooling below the growth temperature first causes the nonbilayer lipids to be transformed into fluid bilayers and mix with the fluid bilayer-forming lipids. This is usually a reversible process in cell membranes because on rewarming the nonbilayer lipids reassociate with membrane components that constrain them into a bilayer configuration. However, further cooling results in the phase separation of the nonbilayerforming lipids into lamellar gel phase under conditions in which the bilayer-forming lipids are in a fluid phase. The permeability-barrier properties and integrity of the membrane are compromised when the membrane is rewarmed because the phase-separated gel phase is able to form nonbilayer structures as it becomes concentrated during formation of the gel phase. The lipid–phase separation model is illustrated by the cartoon in Figure 1.7. Equally damaging lipid–phase separations have been identified in biological membranes that have been subjected to heat stress. Thus chloroplast thylakoid membranes first destack when heated to temperatures higher than 35 ◦ C, and this is associated with dissociation of the light-harvesting chlorophyll–protein complexes of photosystem II from the reaction centre and the phase separation of nonbilayer lipid structures at temperatures above 45 ◦ C [44]. Similar effects have been noted as a result of salt stress, in which there is a loss

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intrinsic protein

(b)

TH > T > TC

T > TH

(d)

(c)

T > TH

TC > T

bilayer-forming lipid non-bilayer-forming lipid

Figure 1.7 branes.

The lipid–phase separation model of low-temperature damage to biological mem-

of photosynthetic functions that can be counteracted by an increase in the proportion of membrane lipids with polyunsaturated fatty acyl residues and the presence of compatible solutes [45]. 1.5.4.2

Membrane Lipid Homeostasis in Homoiothermic Organisms

As has been noted in the above discussion, the lipid composition of the membranes of homoiothermic organisms is highly complex, consisting of hundreds of individual molecular species. Cells have many subcellular organelles which are distinguished not only by the particular proteins but by the mix of lipid molecular species. Clearly, because the identity of each morphologically distinct membrane is preserved within relatively narrow limits, biochemical mechanisms must be in place to sense the membrane composition and to replace lipids that have been oxidized or otherwise damaged. Integral to this is the process of membrane biogenesis. Membranes are not synthesized de novo but are differentiated from existing membrane by insertion of newly synthesized lipids and proteins. With the notable exceptions of egg yolk, lung surfactant and the prolamellar bodies of protoplasts, cells do not synthesize more membrane lipids than they need to satisfy their growth requirements. The primary site of synthesis of membrane lipids is the endoplasmic reticulum; with some classes of lipid required for mitochondria and chloroplasts synthesized in these particular organelles. Membrane buds off from the endoplasmic reticulum to undergo differentiation through the trans-Golgi network, ultimately forming plasma membrane at the cell periphery. Lipids may also be exchanged between different

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membranes by a process mediated by soluble lipid-exchange proteins, of which many specific for particular lipids have been identified. Lipids are also constantly undergoing metabolic turnover: this may be by routes that involve reactions which do not result in products that lose their identity as lipids, for example partial turnover by acyl exchange between lipids. Alternatively, lipids may be degraded to water-soluble products and replaced by new lipids. It can be seen that preserving the huge array of molecular species of lipid in each subcellular membrane of cells is a prodigious task. A significant proportion of the energy consumption of cells is devoted to the biosynthesis of membrane lipids to serve the process. Indeed, the greatest part of the oxygen uptake by most cells is consumed in the desaturation of fatty acids rather than in synthesis of ATP. 1.5.4.3

Membrane Lipid Adaption in Poikilothermic Organisms

When poikilothermic organisms are exposed to changed environmental conditions they respond by changing the lipid component of their cell membranes. This process of adaptation is based on biochemical sensors that detect the physical properties of the lipid bilayer and modulate biosynthetic pathways responsible for retailoring the membrane lipids [46]. The changes in membrane lipid composition resulting from a shift to lower temperatures vary depending on the particular organism but can include one or a combination of the following factors: 1. An increase in the number of unsaturated double bonds in the fatty acyl chains. This is accomplished by the action of fatty acid desaturases, which insert cis-unsaturated bonds at specific locations in the fatty acid. 2. A partial turnover of the membrane lipids in situ. This can involve the retailoring of molecular species of lipid by acyl-exchange reactions. 3. A change in the proportions of the major lipid classes. Some phospholipid classes are enzymaticlly interchangeable, so that the exchange results in generation of new molecular species within each phospholipid class. 4. A shortening of the fatty acyl chains. This can be brought about by shortening existing chains or by de novo synthesis of shorter chains. 5. A change in the type of fatty acid, such as incorporation of branched-chain fatty acids or an alteration in the proportions of iso- and antiiso-branched fatty acids. 6. An overall increase in the rate of lipid biosynthesis, which results in an increase in the lipid : protein ratio of the membrane. Upward shifts in the growth temperature are generally associated with a reversal of the changes that are seen on exposure to low temperatures. Because these adaptive changes involve biochemical reactions, the rate of adaptation is reflected in the extent of the environmental perturbation. Furthermore, strategies have evolved in some poikilothermic organisms, like higher plants, to attenuate the effects of temperature by changing the way the constituents of the membrane are organized. Plants, it must be emphasized, must be able to adapt to diurnal temperature ranges of many tens of degrees, which requires more than biochemical responses.

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1.6 The Action of Antimicrobial Lipids on Cell Membranes Since the physical properties of antimicrobial lipids are those of mild surfactants their primary target is the lipid bilayer matrix of cell membranes. There are no significant differences in the basic structures of membranes of pro- and eukaryotes with respect to the functions of the lipid components of these membranes but the proportion of the major lipid classes and the molecular species of lipid within each of these classes will differ from one membrane type to another. Thus there is currently little evidential basis for predicting how a particular antimicrobial lipid will act to selectively perturb one membrane as opposed to another. The basis of selectivity in the absence of this information is to target the antimicrobial lipid to the organism of interest and protect the host as far as possible by avoiding damaging exposure. There is nevertheless a considerable amount of information about the action of surfactants on membrane lipids that may guide inquiry on the action of weak surfactants, in which antimicrobial lipids are included. The impetus for studies of detergent action on biological membranes comes from the recognition that selective detergent extraction of cell membranes provides a useful method of fractionation of functional domains of the structure. Detergent fractionation relies on the selective solubilization of membrane lipids, suggesting that antimicrobial lipids, like detergents currently employed in membrane fractionation, are targetted to particular lipids of the membrane bilayer matrix. The detergents used in membrane fractionation are of the general type of nonionic surfactants, which comprise of an alkyl chain or other hydrocarbon moiety linked to a polyhydroxylated polar group. Not all are nonionic, as some zwitterionic surfactants, such as CHAPS, have similar properties to those of nonionic surfactants in solubilization of membrane lipids. Amongst the most widely used nonionic detergents for fractionating biological membranes is Triton X-100 [47]. This has a hydrophobic component consisting of octylphenol coupled with polyethyleneglycolether. It is a relatively strong surfactant with a critical micelle concentration of 0.2 mM at 25 ◦ C. Other nonionic detergents, including octylglucoside, Lubrol and Brij96, have been examined for their selective action in solubilizing membrane lipids, and all have been found to be different from one another. From this it may be concluded that while the underlying mechanism of detergent solubilization of membrane lipids may be the same, the patterns of solubilization differ from one detergent to another. This suggests that while the mechanism of action of different antimicrobial lipids on the lipid bilayer matrix of target membranes may be identical they are likely to differ in their perturbation of particular membranes. The example of Triton X-100 is instructive in providing a perspective on how a particular surfactant acts on different membrane lipids because of the considerable body of evidence available on its properties and behaviour. Bilayers of pure phosphatidylcholines are completely solubilized by Triton X-100 at effective detergent : lipid ratios of about 3.5 : 1 when the phospholipid is in the liquid-disordered state. Interestingly, phosphatidylcholines with unsaturated fatty acyl chains are more resistant to solubilization by Triton X-100 than are those with fully saturated chains. Solubilization of phosphatidylcholine with saturated chains in the gel phase appears to be dependent on the length of the acyl chains [48]. Homologues of phosphatidylcholines with diacyl chains C14 and C15 are readily solubilized by Triton X-100 at 20 ◦ C with a detergent : lipid molar ratio of 5 : 1, whereas phospholipid homologues with C16 or longer chains remain completely insoluble under these conditions.

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Fluid bilayers of sphingomyelin are more readily solubilized by Triton X-100 than are those of phosphatidylcholines under the same conditions, requiring detergent : lipid ratios of only about 1.5 : 1 to achieve complete solubilization. However, sphingomyelin in the gel phase is almost insoluble at 20 ◦ C in the presence of Triton X-100. The presence of cholesterol, but not other sterols like androsterol which lack the hydrocarbon tail, reduces the tendency of phospholipids to be solubilized by Triton X-100. This effect has been ascribed to the formation of liquid-ordered phases by interaction of the sterol with the phospholipid. It is noteworthy that androsterol is not able to form liquid-ordered phases with membrane phospholipids [49]. Liquid-ordered phases are more stable in binary mixtures of cholesterol and sphingomyelin. It has been shown that binary mixtures of sphingomyelin with 15 mole% cholesterol are insoluble in Triton X-100 at 50 ◦ C with a detergent : lipid molar ratio of 5 : 1 and insoluble at 22 ◦ C with 20 mole% cholesterol [50]. The selective solubilization of lipids in ternary mixtures of phosphatidylcholine/sphingomyelin/cholesterol by Triton X-100 has been examined in mixtures where the proportions of sphingomyelin and cholesterol were maintained equimolar while the proportion of phosphatidylcholine varied [51]. The results indicated that regardless of the forces that maintain the liquid-ordered structure of sphingomyelin–cholesterol, the lipids became more susceptible to detergent solubilization as the proportion of phosphatidylcholine in the mixture was increased. Such studies are complicated however by the fact that although the proportions of sphingomyelin and cholesterol in the mixture are equimolar, cholesterol also partitions into the phosphatidylcholine domain in proportion to the size of this domain. Also, caution is required in the interpretation of the results of such studies because the Triton X-100 itself may create ordered lipid domains from otherwise homogeneous fluid bilayers formed by lipid mixtures and some attempts have been made to account for this in explaining the selective solubility of fluid bilayer domains [52].

1.7

Conclusions

On the basis of these studies it is likely that antimicrobial lipids, which have a lower critical micelle concentration than Triton X-100 and related detergents, will interact with the less ordered domains of cell membranes. These domains are associated with the maintenance of the permeability-barrier properties and interpolation of intrinsic membrane proteins into the lipid bilayer matrix. Their mode of action on biological membranes in this case is to perturb the selective permeability properties of the target membrane, resulting in dissipation of solute gradients. The influence of such lipids on membrane protein functions cannot be discounted and indeed has been considered as the basis of a new approach to the modulation of cell behaviour [53]. Whether targeting of antimicrobial lipids to disordered lipid domains of prokaryotic membranes can be engineered to be selective is a challenge yet to be met.

Acknowledgements The work was aided by a grant from the Human Frontier Science Programme (RGP0016/2005C).

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47. Goni, F.M., Alonso, A., Bagatolli, L.A. et al. (2008) Phase diagrams of lipid mixtures relative to the study of membrane rafts. Biochim. Biophys. Acta, 1781, 665–684. 48. Patra, S.K., Alonso, A. and Goni, F.M. (1998) Detergent solubilization of phospholipid bilayers by surfactants. Biochim. Biophys. Acta, 1373, 112–118. 49. Gao, W.Y., Chen, L., Wu, R.G. et al. (2008) Phase diagram of androsteroldipalmitoylphosphatidylcholine mixtures dispersed in excess water. J. Phys. Chem. B., 112, 8375–8382. 50. Patra, S.K., Alonso, A., Arrondo, J.L.R. and Goni, F.M. (1999) Liposomes containing sphingomyelin and cholesterol: detergent solubilization and infrared spectroscopic studies. J. Liposome Res., 9, 247–260. 51. Sot, J., Collado, M.I., Arrondo, J.L.R. et al. (2002) Triton X-100-resistant bilayers: effect of lipid composition and relevance to the raft phenomenon. Langmuir, 18, 2828–2835. 52. Keller, S., Tsamaloukas, A. and Heerklotz, H. (2005) A quantitative model describing the selective solubilization of membrane domains. J. Am. Chem. Soc., 127, 11469–11476. 53. Escriba, P.V., Gonzalez-Ros, J. M., Gonui, F.M. et al. (2008) Membranes: a meeting point for lipids, proteins and therapies. J. Cell. Mol. Med., 12, 829–875.

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2 Antibacterial Effects of Lipids: Historical Review (1881 to 1960) Halldor Thormar Faculty of Life and Environmental Sciences, University of Iceland, Reykjavik, Iceland

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

Introduction Antibacterial Activity of Soaps 2.2.1 Antibacterial Action of Fatty Acids and Their Derivatives Inhibition of Lipids and Serum Albumin against the Antibacterial Action of Soaps Diverse Actions of Fatty Acids and Their Salts on Bacteria The Nature of the Bactericidal Action of Fatty Acids A Possible Role of Soaps and Fatty Acids in Host Defence against Bacteria Studies of Prophylactic and Therapeutic Applications of Soaps and Fatty Acids Conclusions

25 26 29 33 34 36 38 40 43

2.1 Introduction In 1881, Robert Koch [1] wrote a report on disinfection, which was based on a number of experiments, particularly on the effect of various chemical compounds on Bacillus anthracis. A few years earlier he had shown that this bacterium is the cause of anthrax, by eliciting the disease in experimental animals injected with the bacterium grown in pure culture. Thus, the anthrax bacillus was the first bacterium conclusively demonstrated to be the cause of a disease. This discovery made it possible to test the effects of various disinfectants in vitro, not only against the antrax bacillus but also against a number of other Lipids and Essential Oils as Antimicrobial Agents
C 2011 John Wiley & Sons, Ltd

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bacterial species which in the following years were shown to be the causes of well-known diseases, such as cholera, typhoid fever, tuberculosis and diphtheria. On page 271 in his report on disinfection, Koch drew attention to an observation which suggested that certain fatty acids inhibited bacterial growth. However, he could not confirm this observation by further experiments, which showed that neither butyric acid nor oleic acid in 1 : 2000 dilution had an effect on the growth of anthrax bacillus. On the other hand, potassium soap in 1 : 5000 dilution had an inhibiting effect and in 1 : 1000 dilution (0.1%) completely stopped the growth of the anthrax bacillus. Since free potassium had a much smaller effect, Koch concluded that a different component of the soap he used, most likely some kind of fatty acid, caused the inhibition of bacterial growth. This was the first indication that lipids might have an antimicrobial effect and that ordinary soaps, which are potassium or sodium salts of fatty acids, could be used as disinfectants as well as for cleaning – a function that had been known for thousands of years. In the following years, Koch’s discovery led to numerous studies on the antimicrobial effects of various types of soap on a variety of pathogenic bacteria, which at the end of the nineteenth century caused serious illnesses in the Western world, but are now a problem only in Third World countries. The early studies of the effect of soaps on bacteria were largely of a practical nature; that is, they sought to determine to what extent soaps could be used for disinfection under various conditions. However, there was also an early interest in the question of which components of soaps were the active ingredients. After it had been established that the antibacterial activity resided in the fatty acid moiety, studies were done on the mechanism by which soaps either inhibit the growth of bacteria or kill them. There was even an interest in a possible therapeutic use of soaps or fatty acids and in whether or not fatty acids and their salts found in body tissues might play a role in natural defence against bacterial infection. With the advent of antibiotics in the early 1940s the interest in fatty acids and other lipids as antibacterial agents decreased greatly, at least temporarily, but there was a new interest in their effect on viruses and particularly on fungi. Fungicidal dosage forms containing fatty acids or salts thereof were formulated for use against fungal infections, particularly of the skin and mucosal membranes.

2.2 Antibacterial Activity of Soaps Following the report by Koch in 1881 on the antibacterial effect of potassium soap [1], a considerable effort was made to test the possible germicidal effect of different types of soap against pathogenic bacteria and thus their potential as general disinfectants. These early studies were reviewed by Reithoffer in 1896 [2], Serafini in 1898 [3] and Konr´adi in 1902 [4]. According to these authors, the results of Koch on the anthrax bacillus were confirmed by Max Kuisl in 1885, but in his hands the potassium soap in 5% concentration had no inhibiting effect on typhoid or cholera bacteria. Furthermore, they quoted studies by Di Mattei from 1889, who tested sodium soaps against four different species of bacteria. In contrast to Kuisl, Di Mattei found that soaps killed cholera bacteria (Vibrio cholerae) in culture medium within minutes to hours, whereas it took longer to kill typhoid and anthrax bacilli, and Staphylococcus pyogenes aureus isolated from pus survived for at least 24 hours. In 1890, Behring [5] studied the effect of about 40 different commercial soaps on the anthrax bacillus and concluded that their activity depended on their free alkali contents.

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In 1893, Jolles [6] tested the activity of five different soaps against the cholera bacterium in bouillon culture and found that all five soaps, in 8 to 10% solution, killed the bacteria within one to two minutes at 15 ◦ C, as determined by plate culture of samples taken at different times. At decreasing soap concentration it took increasingly longer time to kill the bacteria, but the killing time decreased with increasing temperature. He found very little difference in the germicidal activities of the five soaps against the cholera bacilli. In a study published in 1895, Jolles [7] tested the activity of a potassium soap against typhoid and coli bacilli in bouillon culture. He found that a 6% soap solution killed the typhoid bacteria within 15 minutes at 4 to 8 ◦ C and the killing time increased with decreasing soap concentration. The killing time was longer at 18 ◦ C than at lower temperatures. The coli bacteria were more resistant than the typhoid bacilli and were killed in one hour by a 5 to 7% soap solution at 4 to 8 ◦ C. The killing time decreased with increasing soap concentration, but was slightly longer at 18 and 30 ◦ C than at the lower temperatures. Using a recent isolate from a fatal case of cholera, Nijland in 1893 [8] tested the killing of cholera bacilli in water by a number of different commercial soaps, either white hard sodium soap or green soft potassium soap. He found that the potassium soap was more active than the sodium soap, totally killing the bacteria in the water in 10 minutes at a concentration of 0.24%. In 1896, Beyer [9] tested a number of commercial soaps with various free alkali contents against cholera bacilli, typhoid bacilli, Bacterium coli, S. pyogenes aureus and diphtheria bacilli, all recent isolates from patients. The soaps were tested in a concentration of 3% at various temperatures. They were found to differ in their germicidal activities but, in contrast to Behring’s results, showed no correlation with the free alkali contents of the soaps. Reithoffer [2] pointed out the conflicting results of previous studies on the germicidal activity of soaps, which he considered to be partly caused by faulty test methods, such as adding the soap solutions to bacteria in bouillon culture. He therefore used a standardized protocol to study the activity of three different commercial soaps, in which a 24-hour bacterial culture was suspended in sterile distilled water and then filtered to remove clumps of bacteria. An equal volume of a soap solution was then added to the bacterial suspension and mixed at room temperature. Samples were removed from the mixture at intervals, inoculated into sterile bouillon and then regularly passed into a second bouillon. The bouillon cultures were incubated for eight days at 37 ◦ C. A 10% soap solution killed cholera bacilli within half a minute under these experimental conditions. Sodium or potassium hard soaps1 in 2% solutions killed the bacteria within one minute, whereas the killing time for a 2% solution of potassium soft soap2 was two to five minutes. The potassium hard soap was even highly germicidal in 1% solution, killing the bacteria in less than one minute. The killing time was five minutes for a 0.5% soap solution and then increased rapidly at lower concentrations so that a 0.1% solution of potassium hard soap did not kill all the bacteria in two hours. A different strain of cholera bacilli isolated in Africa (Massaua) was also killed by low concentrations of soap, but in a slightly longer time. Thus, Reithoffer concluded that soaps are highly germicidal against cholera bacilli. The same method was used to test the germicidal activity of the soaps against typhoid bacilli and B. coli. The activity against these bacteria was found to be less than against cholera bacilli and a 10% concentration 1 Hard soaps include 2 Soft soaps are of a

a variety of soaps of different ingredients, usually sodium soaps, and are hard and compact. green or brownish yellow color and of semifluid consistence. They are made from potash or the lye from wood ashes. They are strongly alkaline and often contain glycerin.

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was needed to kill the bacteria within one minute. As for cholera bacilli, the potassium hard soap was the most active and killed within three minutes in a 5% solution. On the other hand, the three soaps were almost without germicidal activity against S. pyogenes aureus, even in a 20% solution for one hour. Serafini [3] studied the germicidal activity of a number of commercial soaps against the Massaua strain of cholera. In his experiments, 1 ml of a 24- to 48-hour bouillon culture was added to 10 ml of sterile soap solution in either distilled or tap water and the mixture was kept at 10 to 15◦ C. Samples were removed at intervals and inoculated into culture tubes with bouillon culture medium. The different soaps, both hard sodium soaps and soft potassium soaps, were found to kill the bacterium, the killing time decreasing from a few hours to a few minutes when the soap concentration increased from 0.25% to 5%. There was, however, a distinct difference in the germicidal activities of the soaps. Serafini speculated about the possible cause for this difference and, therefore, which constituent of the commercial soaps was responsible for their germicidal activity. Based on his results he concluded, in agreement with Beyer, that the active ingredient was not free alkali but rather the neutral sodium and potassium salts of fatty acids – that is, the soap proper – as also suggested by Koch [1]. However, based on his own results and the results of others, Serafini could not detect a difference in the germicidal activities of sodium and potassium soaps. On the other hand, Konr´adi [4] in 1902 concluded, after studying a commercial perfumed soap, that its germicidal activity against anthrax spores was due to an odorous essential oil, terpineol, not to salts of fatty acids. In 1908, Reichenbach [10] addressed the question of the conflicting results of studies on germicidal activity of soaps and claimed that they were not due to different research methods but rather to a difference in the composition of the commercial soaps used by the various investigators. He emphasized the importance of determining separately the germicidal activities of the main components of commercial soaps, that is 1) salts of fatty acids, 2) free alkali and 3) other additives, and he was the first to carry out a systematic work with chemically pure compounds. For his studies, Reichenbach synthesized the potassium salts of purified fatty acids, which were commonly found in soaps, and tested each salt against B. coli. A 24-hour culture of the bacterium was suspended in water. After filtering, three drops of the suspension were added to 10 ml of a soap solution, which was thoroughly mixed, and samples were removed at intervals and inoculated into culture medium. Of the salts of saturated fatty acids, the potassium palmitate showed the highest activity, a 0.72% concentration killing the bacteria in less than five minutes. Stearate and particularly myristate were somewhat less active, and the activities of salts of shorter saturated fatty acids decreased with the chain length. In contrast, potassium salts of long-chain unsaturated fatty acids showed very little germicidal activity against B. coli, even at concentrations as high as 13 to 14%. From these results, Reichenbach concluded that the difference in activity of individual potassium salts against B. coli partly explained the difference in activity of various commercial soaps, which are made from a variety of fatty acids. Reichenbach also separated free alkali from soaps, which was mostly in the form of carbonates, and tested the activity against B. coli. In agreement with the results of Serafini [3], the free alkali could not explain the germicidal activity of soaps. The same was true of additives such as terpineol, although they were found to have a slight activity against typhoid bacilli. In all studies of the germicidal action of soaps, care was taken to prevent carry-over of residual germicidal activity from the bacterial soap mixtures to the assay medium, such as

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bouillon, by dilution of the soap mixture. Geppert in 1889 [11] first drew attention to the false results that might be caused by such carry-over and advised the use of a neutralizing agent to stop the action of a disinfectant. 2.2.1

Antibacterial Action of Fatty Acids and Their Derivatives

Following the work of Reichenbach [10] on the bactericidal action of salts of purified fatty acids, studies were increasingly focussed on the fatty acids, which are commonly found in soaps, and their derivatives. In the literature, the salts of fatty acids were commonly referred to as ‘soaps’. In 1911, Lamar [12] reported the results of a study of the action of sodium oleate on a virulent strain of pneumococcus. When sodium oleate was added to a broth culture of the pneumococcus in 0.5 to 1% concentration the diplococci were killed within 15 to 30 minutes. Higher dilutions of the sodium oleate were less active and 0.1% solutions did not kill the pneumococci in one hour. In an extension of the previous study, Lamar [13] tested the haemolytic activity of a number of free fatty acids and their potassium and sodium salts. He used a mixture of unsaturated fatty acids, mostly oleic and linoleic acids, extracted from the livers of rabbits poisoned with phosphorus, and also several single isolated acids from other sources, including oleic, linoleic and linolenic acids. All of the unsaturated fatty acids were strongly haemolytic in 1 : 20 000 to 1 : 40 000 dilution and caused a complete lysis of red cells from dogs in one hour. The potassium and sodium salts were haemolytic in a higher dilution than the corresponding acids, with the highest acivity shown by potassium linolenate in a 1 : 150 000 dilution. The potassium salts were tested against pneumococci in the same way as previously reported for sodium oleate [12]. There was a close relationship between their germicidal activity against virulent pneumococci and their lytic activity for red blood cells. Potassium linoleate and linolenate were the most germicidal, being four and six times as destructive as sodium oleate. Nichols [14] tested the bactericidal activity of chemically pure sodium salts of oleic and stearic acids and of brown soap, which is commonly used for dish washing. He found that a 0.5% solution of sodium oleate killed Streptococcus pyogenes, Bacillus influenzae and pneumococci in less than half a minute at room temperature, whereas Staphylococcus aureus and typhoid bacilli were not killed in 10 minutes. In contrast, sodium stearate had no or very little germicidal effect on any of the bacteria tested. Brown soap had a similar germicidal effect as the oleate, although it took two minutes to reduce the number of streptococci from innumerable to 15. The bactericidal activity of the soaps was dependent on the pH and was lost if it fell from about pH 8.5 to 7 by addition of acid, probably due to precipitation of the soap from solution. Walker, in a series of papers [15–18], studied the germicidal properties of chemically pure soaps. He emphasized the practical importance, from a hygienic point of view, of knowing which soaps are germicidal against the different pathogens. In addition he pointed out other aspects which make studies of fatty acids and their salts worthwhile, namely their possible role in the defence of the body against bacteria and their potential as therapeutic compounds, for example in treatment of wounds. Potassium and sodium salts were made from a series of chemically pure fatty acids and diluted in distilled water. Bacteria in broth culture were added to the soap solution at 20 ◦ C and samples were taken after 2.5 and 15 minutes and tested for viability by inoculation into

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broth. The soaps were tested against typhoid bacilli, haemolytic streptococci, pneumococci and staphylococci. They were found to vary markedly between themselves and with regard to their germicidal activity against the different bacteria. Of the soaps of saturated fatty acids, laureate was the most active, a 0.002% solution killing the pneumococci in 15 minutes. The shorter- and longer-chain saturated soaps were less active. Among the 18carbon unsaturated soaps the activity increased with the number of double bonds, from oleates to linolenates. There was no or very little difference in the activities of sodium and potassium salts, indicating that the germicidal activity resides in the fatty acid moiety. The pneumococci were the most susceptible to the germicidal activity of all the soaps and the streptococci were somewhat less sensitive. The typhoid bacilli were not killed by the unsaturated soaps and only by low dilutions of the saturated soaps, of which 0.7% palmate was the most active. The staphylococci were resistant to all of the soaps. These results [15] were therefore in agreement with those of Nichols [14]. Of the soaps studied, sodium and potassium laurates seemed to have the most general germicidal activities, since they killed pneumococci and streptococci in high dilutions and also had an appreciable activity against typhoid bacilli, which might be sufficient to prevent the spread of the bacteria and thus be of hygienic value. Addition of serum, and to a lesser extent of broth, was found to inhibit the germicidal activity of soaps, as demonstrated by the effect of serum on the activity of sodium laurate against pneumococci. The serum effect was less pronounced in the case of typhoid bacilli. In contrast to the results of Jolles [7], it was found that the germicidal activity of soaps increased with temperature. In a paper published in 1925 [16], Walker reported a study of the germicidal properties of commercial soaps as well as of neutral soaps prepared in the laboratory from known fats and oils. The experiments were carried out in the same way as in the previous study and the same bacteria were used, except that diphtheria bacilli were tested instead of pneumococcci. Since all of the commercial soaps contained mixtures of different fatty acids they showed similar activities against each type of bacterium, although coconut-oil soap was the most active against the typhoid bacillus at 20 ◦ C. On the other hand, there was a distinct difference in the activities of the pure soaps against the different germs. The diphtheria bacillus was the most susceptible, similar to pneumococci, followed by the streptococci. The typhoid bacillus was much more resistant and S. aureus was not killed by any of the soaps under the experimental conditions. All the soaps were more germicidal at 35 ◦ C than at 20 ◦ C. Sodium laurate and potassium palmitate were tested against coli bacillus and found to be slightly less active against this bacterium than against the typhoid bacillus. Both types of bacterium were resistant against sodium oleate and sodium linolate at 20 ◦ C for 15 minutes. However, high concentrations of coconut-oil soap manufactured in the laboratory killed the coli bacilli within one minute and a commercial coconut-oil soap within 2.5 minutes. The germicidal activity of coconut-oil soaps against typhoid bacilli and to a lesser degree against coli bacilli seemed to be due to their high contents of saturated fatty acids, particularly lauric acid, and very low amounts of unsaturated acids. In a later publication [17], Walker extended his studies of soaps to include meningococcus and gonococcus. He found both cocci to be susceptible to chemically pure soaps of both saturated and unsaturated fatty acids tested at 20 ◦ C for 2.5 and 15 minutes. Since meningococcus was somewhat less susceptible than gonococcus to the germicidal action of all the chemically pure soaps, its susceptibility to four common commercial soaps was also tested. As expected, the meningococcus was killed by all of the soaps in 2.5 minutes

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at 20 ◦ C at a soap concentration of approximately 0.04%. Chemically pure soaps were also tested against dysentery and paratyphoid bacilli. Only soaps of saturated fatty acids were found to kill these intestinal bacteria and their germicidal activities were almost identical to those previously found for the typhoid bacillus. A commercial soap prepared from coconut oil, and therefore mostly containing salts of saturated fatty acids, was found to kill these intestinal bacilli in 15 minutes at 20 ◦ C in a concentration of approximately 0.4 to 1.6%. From these studies, Walker [17, 18] concluded that the bacteria he had studied could be placed into two groups. One group, which included pneumococci, gonococci, meningococci, streptococci and diphtheria bacilli, was highly susceptible to sodium and potassium salts of both saturated and unsaturated fatty acids and was readily killed by any ordinary soap. In the other group were the enteric bacteria, that is typhoid, paratyphoid, dysentery and coli bacilli, which showed a moderate susceptibility to salts of saturated fatty acids. They were only killed by soaps which contained these salts, such as coconut-oil soap, but were completely resistant to soaps of unsaturated acids at ordinary temperatures. Following the study by Nichols [16], who found that the sodium salt of oleic acid was most germicidal at alkaline pH, Eggerth in 1926 [19] did a thorough study of the effect of pH on the germicidal activity of chemically pure potassium soaps on a series of fatty acids. Serial dilutions of the soaps were made in distilled water and the pHs of the mixtures of bacteria and soap were controlled by buffers ranging from pH 3.8 to 10.0. The soap solutions were tested against Streptococcus pyogenes, S. aureus, V. cholerae, Bacillus diphtheriae and Bacillus typhosus. The tests were performed by inoculating samples of bacterial broth cultures into the appropriate buffer, then adding the soap solutions. The mixtures were incubated at 37 ◦ C and samples were removed at intervals ranging from 30 minutes to 18 hours and inoculated on to agar plates. The somewhat complicated data emerging from this study showed the germicidal activity of soaps to vary not only with the pH, but also with their chain length and saturation, and with the species of bacterium studied. Generally it was found that the short- and medium-chain saturated soaps were most active in acid solution and the long-chain soaps at alkaline pH, although the point of change varied with the bacterium. From the generally higher activity of the short- and medium-chain soaps at acid pH it was concluded that the corresponding fatty acids were more germicidal than their potassium salts. It was considered likely that the low activity of long-chain soaps at acid pH was due to the diminishing solubility of fatty acids with increasing chain length and therefore that their germicidal activity failed because they were not soluble in the needed concentration. On the other hand, when a fatty acid was sufficiently soluble it was more active than the corresponding soap. It was suggested that the greater germicidal activity of fatty acids might be due to the fact that they were less dissociated than their salts and could therefore penetrate more easily into a bacterium. Dresel in 1926 [20] reported a study on the germicidal action of a number of chemically pure fatty acids on both Gram-positive and Gram-negative bacteria. Emulsions of fatty acids were diluted into bouillon cultures and samples were removed at intervals and tested for bacterial viability. The medium-chain fatty acids showed the highest germicidal activity against Gram-positive bacteria, such as diphtheria, anthrax bacilli, streptococci, pneumococci and staphylococci, but had no activity against most Gram-negative bacteria, such as typhoid and coli bacilli. A microscopic examination of the killed bacteria showed that the fatty acids caused a complete lysis of the bacterial cells.

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Hettche in 1934 [21] compared the bactericidal effects of a number of chemically pure sodium salts of fatty acids against both Gram-positive and Gram-negative bacteria. He found that 0.1% sodium salts of palmitic and oleic acids had no effect on coli bacilli in bouillon. Staphylococcus was tested in the same way with sodium salts of fatty acids. The salts of capric, stearic and palmitic acids had little or no effect on the bacterium, whereas 0.1% sodium oleate caused a 10-fold reduction in viable bacteria in one hour and linoleate and linolenate killed all the bacteria. When tested in lower concentrations, linolenate killed the highest number of bacteria. The bactericidal activity therefore increased with the number of double bonds in the 18-carbon chain, as did the haemolytic activity. In 1934, Barnes and Clarke [22] determined the minimum amounts of sodium ricinoleate and oleate needed to kill pneumococci of types I, II and III in broth culture. Series of dilutions of the two soaps were inoculated with broth cultures and incubated at 37.5 ◦ C. Microscopic examination and subcultures of samples taken after incubation for 24 hours showed that 0.0004% oleate and 0.004% ricinoleate were effective in killing all three types under these conditions. This study was done with the aim of determining the concentration of soap which would kill the bacteria without impairing the bacterial antigens needed for preparation of a vaccine. Bayliss in 1936 [23] studied the germicidal action of a number of sodium soaps and other chemicals at pH 8.0 against four different bacteria, S. aureus, Streptococcus lactis, Escherichia coli and Diplococcus pneumoniae. Of the saturated soaps, sodium myristate was found to be most active against pneumococci, with sodium laurate second in activity. Soaps of unsaturated long-chain fatty acids were much more active than the corresponding saturated soaps against pneumococci. The activity was not found to increase with the number of double bonds but the positions of the double bonds markedly affected the germicidal activity. As in the case of pneumococci, myristate and laurate were active in killing S. lactis, and sodium oleate was equally active. In contrast to pneumococci, the presence of a second double bond, as in linoleate, increased the activity. Addition of a hydroxyl group, as in ricinoleate, enhanced the activity against the streptococcus but decreased the activity against pneumococcus. The effectiveness of most soaps against the staphylococci was very low, sodium undecylenate being the most active of the soaps tested. The results for E. coli were similar to those for staphylococci, with sodium undecylenate being the most active soap. Thus, the bacteria showed a greatly variable susceptibility to the different soaps. Bayliss [23] stated that ‘studies on the properties of soaps are of importance because of their common use as detergents, in the preparation of vaccines, in the treatment of disease and because of their natural occurrence in the animal body where they may have some effect in determining the resistance of the animal organism to infection’. In 1941, Cowles [24] studied the bactericidal action of the lower fatty acids, from acetic to caprylic acids, against S. aureus and E. coli at low pH to ascertain the relative importance of the hydrogen ion and the undissociated fatty acid in this action. The results showed that at pH above about 2.6 the hydrogen ion lost its bactericidal activity against S. aureus, and at pH above 1.75 the activity was lost against E. coli. For E. coli, the bactericidal action seemed to be due almost entirely to the undissociated acids. For S. aureus, the action of the lowest acids, for example acetic acid, seemed to be partly caused by hydrogen ions, whereas the action of the higher acids, caproic and caprylic acids, was largely due to the un-ionized molecule. The anions did not seem to play a significant role in the bactericidal action.

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Weitzel in 1952 [25] tested the effect of a large number of fatty acids in low concentrations on long-term growth of tubercle bacilli in culture medium. Of the saturated fatty acids, the medium-chain undecylic and lauric acids were the most active. The unsaturated fatty acids studied were mostly of 18-carbon chain length. Oleic acid and other 18-carbon acids with one double bond of different configurations showed similar activities, which were not different from the saturated stearic acid. Linoleic acid and other 18-carbon fatty acids with two double bonds were distinctly more active than either oleic acid with one or linolenic acid with three double bonds. This did not agree with the results from other studies on tubercle bacilli, which found the activity of unsaturated fatty acids to increase with the number of double bonds. Free fatty acids from chaulmoogra oil showed by far the highest activity as compared with free fatty acids extracted from various other natural sources, such as linseed oil and cod-liver oil, although they had higher contents of fatty acids with a large number of double bonds. The high antitubercular activity of chaulmoogra oil was most likely due to its content of the 18- and 16-carbon cyclopentenyl fatty acids, chaulmoogric and hydnocarpic acids.

2.3 Inhibition of Lipids and Serum Albumin against the Antibacterial Action of Soaps As shown by Noguchi [26], Landsteiner and Ehrlich [27], Lamar [12] and Walker [15], serum inhibits the germicidal activity of soaps. Eggerth in 1927 [28] studied the inhibiting effect of serum at various pHs. As in his previous study [19], the soaps were made from chemically pure fatty acids and tested against the same bacteria, except for V. cholerae, and the experiments were carried out in the same way, either without serum or with sheep serum in a series of dilutions. In general, addition of serum reduced the activity of soaps at all pH values tested, the activity becoming gradually lower with increasing serum concentration. This was illustrated in mixtures of streptococci with either myristate or oleate, where the serum inhibited the soap almost equally at both high and low pH. A combination of S. aureus with laurate showed a somewhat different pattern. Here the activity of the soap rose steeply when the pH of the control buffer solution was lowered from pH 9 to 5. The addition of 1 and 5% of serum had no effect on the germicidal titre at pH 8 and 9, but greatly reduced the titre at lower pH, thus flattening the curve. In an attempt to explain the mechanism of serum inhibition, Eggerth tested a number of protein substances not found in serum, such as crystalline egg white, gelatin and casein, in streptococcus–oleate and staphylococcus–laurate mixtures at various pHs and found them all to be inhibitory. However, the germicidal curves over the pH range were different from those with sheep serum, suggesting a different action. On the other hand, serum with or without calcium gave identical results, ruling out the role of calcium in the serum. Emulsions were made of cholesterol, lecithin and other lipoids,3 which were either extracted from serum or prepared from other sources. Such emulsions, particularly those containing lecithin, were found to inhibit the action of soaps against bacteria and gave curves which were identical to those of serum. Lipoid emulsions were soluble in casein solutions and such mixtures were more inhibitory to soaps than casein

3 Lipoids

include fats, their derivatives (e.g. lecithin) and their components (e.g. fatty acids and cholesterol) [28].

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alone. Eggerth concluded that the inhibitory effect of serum on soaps was a complex action of proteins and lipoids which was probably due to adsorption or interactions of soaps with these substances, thus reducing the germicidal activity of the soaps. In 1947 Dubos [29] studied the effect of lipids and serum albumin on the growth of bacteria in liquid media. The growth of human tubercle bacillus was completely inhibited by 0.0001% oleic acid but not by methyl oleate. The effect of oleic acid was completely neutralized by the addition of crystalline serum albumin. The inhibiting effect of serum albumin seemed to be due to formation of a lipoprotein complex between the fatty acid and the albumin. This was supported by the fact that a cloudy fatty acid emulsion became clear upon addition of an adequate amount of serum albumin and at the same time became inactive against bacteria. Of many proteins tested, only native serum albumin had this property. In a following paper, Davis and Dubos [30] studied the interaction between fatty acids and serum albumin. They found that the albumin was able, over a wide concentration range (0.1 to 1.6%), to bind 1 to 2 % of its weight of oleic acid. The binding was firm enough to neutralize the bacteriostatic effect of the fatty acid against tubercle bacillus in liquid medium. Serum albumin was also found to protect sheep red blood cells from haemolysis by oleic acid. Tween 80 (polyoxyethylene sorbitan monooleate) had the same protective effect. Serum albumin was found to have a greater capacity for tightly binding long-chain fatty acids than any other substance tested, such as human serum globulin which had no binding effect. It was speculated that a specific configuration of amino-acid residues on the surface of albumin molecules led to the interaction with fatty acids. A positively charged quaternary nitrogen atom on an amino acid, such as lysin, would be likely to bind to the negatively charged carboxyl ion on the fatty acid. Similarly, a properly located nonpolar residue on an amino acid, such as leucin, might attract the nonpolar chain of the fatty acid by van der Waals forces and thus contribute to a firm binding to the albumin molecule. The affinity of serum albumin for chemotherapeutic agents with anionic groups was considered to be of practical importance.

2.4

Diverse Actions of Fatty Acids and Their Salts on Bacteria

In a study by Avery in 1918 [31], addition of a solution of 0.1% sodium oleate was found to stimulate the growth of B. influenzae on blood agar plates, as well as in oleate–haemoglobin bouillon, whereas the oleate completely inhibited the development of pneumococci and streptococci in the same medium. This study demonstrated a strong difference in the effects of a lipid, namely sodium oleate, on different bacteria. The diverse actions of lipids on various bacteria were later shown in a number of publications and reviewed by Nieman in 1954 [32]. Kodicek and Worden in 1945 [33] studied the effects of long-chain fatty acids on the growth and acid production of Lactobacillus helveticus. Linolenic, linoleic and to a lesser degree oleic acids were inhibitory when added to the growth medium in a concentration of 160 ␮g per 10 ml medium, whereas stearic and palmitic acids stimulated the acid production of the bacterium. The inhibitory action of the unsaturated fatty acids on growth and acid production was reversed by addition of lecithin, cholesterol and a number of other compounds to the medium. Their action was therefore bacteriostatic rather than bactericidal.

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Linoleic acid was found to exert an inhibitory effect on a number of other Gram-positive bacteria but not on Gram-negative bacteria tested, such as E. coli. In a later work, Kodicek [34] studied the bacteriostatic effect of low concentrations of free fatty acids on Gram-positive bacteria, particularly Lactobacillus casei. Of the saturated acids, the lauric acid depressed the growth of this bacterium but the ethyl ester had no effect. The growth-inhibiting activity of the unsaturated fatty acids increased with the number of double bonds and was limited to the naturally occurring cis-forms of the free acid. As in the case of lauric acid, esterification abolished the activity of the acid but unlike with lauric acid, the effect of the unsaturated fatty acids was reversed by cholesterol. In addition to cholesterol, other surface-active agents, especially lecithin, abolished the bacteriostatic effect of unsaturated acids. Humfeld in 1947 [35] observed antibiotic activity in culture medium containing wheat bran and hypothesized that it was due to hydrolysis of fatty constituents of the medium caused by cultured microorganisms. Potassium salts of fatty acids extracted from wheat bran inhibited the growth of S. aureus, Streptococcus faecalis and Micrococcus conglomeratus, similarly to the inhibition caused by potassium linoleate. Potassium salts extracted from the wheat bran stimulated the growth of E. coli in the medium, in contrast to their inhibitory effect on the other bacteria tested. Foster and Wynne in 1948 [36] studied the effect of 10 saturated and unsaturated fatty acids on spore germination of the anaerobic Gram-positive bacterium Clostridium botulinum and found that oleic, linoleic and linolenic acids were strongly inhibitory in germination medium. Curiously, the degree of inhibition depended on the lot of medium used, and starch neutralized the inhibitory action of the fatty acids in medium. The inhibition was specific for the germination process and fatty acids had no effect on the growth of vegetative cells after germination. The germination of other clostridium species tested was less inhibited by the fatty acids than the germination of C. botulinum, and the germination of endospores of aerobic bacilli was not inhibited by the fatty acids. Notably, oleic acid at the highest concentration studied did not kill the spores of C. botulinum in distilled water, and apparently showed inhibitory action only in concert with other inhibitors in the medium. Interestingly, oleic acid had previously been found to be an essential growth factor for Clostridium tetani as well as for the diphtheria bacillus [37]. Hardwick and coworkers in 1951 [38] studied factors in peptone medium which inhibited sporulation of various aerobic bacilli, such as Bacillus mycoides. By fractionation of the medium the antisporulation factors were identified as saturated fatty acids, most of the activity being present in the nonvolatile acid fraction. By comparing the antisporulation activities of pure fatty acids, capric (C10), lauric (C12), tridecylic (C13) and myristic (C14) acids were found to be the most active, whereas the low-carbon-chain volatile C1 to C7 fatty acids were much less active. The activity of saturated fatty acids with 16- and 18-carbon chains was also lower, whereas unsaturated long-chain fatty acids had considerable activity, particularly linoleic and ricinoleic acids. Oleic acid, on the other hand, had little antisporulation activity. All of the saturated fatty acids studied inhibited vegetative growth of the bacilli to various extents, but at concentrations well above those exhibiting antisporulation activity. The unsaturated acids, linoleic and ricinoleic acids, were equally active in inhibiting vegetative growth and sporulation of these aerobic bacilli. Nieman [32] reviewed the literature on the influence of small quantities of fatty acids on microorganisms, particularly bacteria. He discussed the remarkable fact that fatty acids,

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particularly unsaturated at very low concentrations, are able to either stimulate or inhibit the growth of microorganisms. This ‘double effect’ or ‘dualism’ was most clearly demonstrated by the fact that the effect of a fatty acid as a growth factor could be changed into an inhibitory action by increasing its concentration above a critical level. The inhibitory action of low concentrations of fatty acids in the 5 to 50 ppm (parts per million) range, or even lower, had been known for a long time, particularly for Gram-positive bacteria. This inhibitory action, demonstrated by depression of either respiration or growth rate, was in many cases reversible and can therefore be characterized as bacteriostatic. It was influenced by both fatty acid structure and concentration, as well as by the bacterial species. As reported by many investigators, the inhibitory properties of fatty acids are generally most pronounced for longer-chain unsaturated acids. This was particularly true for bacteria. However, saturated fatty acids have also been found to act as growth inhibitors, especially acids with chain lengths around 12 carbon atoms. Dubos had found in 1948 [29] that fatty acids, both saturated and unsaturated, had a bacteriostatic as well as bactericidal effect on tubercle bacilli in protein-free medium. Stearic acid was the least active and the 18- and 22-carbon unsaturated fatty acids were the most active, even in concentrations as low as 0.00 001 to 0.0001%. The bacteriostatic/bactericidal activity of the fatty acids was very much decreased or completely abolished by addition of serum albumin. However, the effect of the fatty acids depended greatly on the experimental conditions, such as the composition of the medium and the size of the bacterial inoculum. Small concentrations of some fatty acids were even found to stimulate bacterial growth under certain conditions, for example when their bacteriostatic effect was inhibited by serum albumin. These studies emphasized the importance of distinguishing between a reversible bacteriostatic (growth-inhibiting) effect and an irreversible bactericidal (killing of bacteria) effect.

2.5 The Nature of the Bactericidal Action of Fatty Acids Several studies have addressed the question of how fatty acids and soaps kill bacteria. In a series of publications [39–41], Eggerth studied the germicidal/bactericidal activity of various soap derivatives with the purpose of throwing light on the relationship between their chemical structure and their germicidal action. In the first study, a hydrogen atom was replaced with bromine on the carbon atom next to the carboxyl group of the fatty acid chain, thus making an alpha-brom fatty acid, which was then changed into the corresponding soap by addition of potassium hydroxide. The brominated soaps of saturated fatty acids were much more soluble in water than the unsubstituted soaps and were generally more germicidal and haemolytic. This was particularly true for soaps of long-chain saturated fatty acids. Thus, potassium palmitate had no activity against S. aureus, whereas the bacterium was killed by bromopalmitate in high dilutions. All of the Gram-positive bacteria tested were most effectively killed by soaps of 16 or 18 carbon atoms and the Gram-negative bacteria by soaps of 12 or 14 carbon atoms. These findings were explained by the hypothesis that the germicidal activity of the soaps for all species of bacteria increases rapidly with the length of the fatty acid chain. However, the ability to penetrate the limiting membranes of the bacteria decreases with equal rapidity, the Gram-negative bacteria being less permeable

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than the Gram-positive. The point of maximal germicidal activity would then be determined by the permeability of bacterial membranes to soap molecules. With low permeability, only soaps with smaller molecules can enter and the maximal activities will be low, whereas with high permeability the longer-chained soaps can act, consequently with higher maximal activities [39]. Like the unsubstituted soaps, the brominated soaps were more germicidal at acid than at alkaline pH, strengthening the previous conclusion that fatty acids are more germicidal than the corresponding soaps [28, 39]. In a study of alpha-hydroxy soaps [40], Eggerth saw a similar change in germicidal activity, compared to unsubstituted soaps, as that observed for the alpha-brom soaps; that is, a shift of the maximal germicidal activities towards longer-chain soaps. However, the germicidal activity of alpha-hydroxy soaps varied greatly with the different bacteria tested, which prompted the author to suggest that they could be used to separate mixtures of bacteria by selective germicidal action [40]. In a paper in 1931 [41], Eggerth pursued the idea of selective action of soaps in a study of the germicidal action of alpha-mercapto and alpha-disulfo soaps. Similar to alpha-brom and alpha-hydroxy substituted soaps, the germicidal activities of these soaps increased rapidly with the length of the fatty acid chain until a maximum was reached, and then fell off. This was taken to support the previous hypothesis that the germicidal action increases with the molecular weight of the soap up to a point where the molecule is too large to penetrate the outer membrane of the bacterial cell. Like the previously studied alpha-substituted soaps, the alpha-mercapto and alpha-disulfo soaps showed a highly selective action, with no two bacterial species having exactly the same action profile. Hotchkiss [42], in a review of the bactericidal action of surface-active agents, proposed the following scenario: the first stage of the interaction is a combination of the ionic group of a hydrophobic–hydrohilic surface-active molecule, such as a fatty acid, with oppositely charged ionic groups on the bacterial surface. As pointed out by A.W. Ralston in a discussion following the review, the surface-active ions might take the form of ionic micelles, which are the highest-charged particles in an aqueous collodial system. The ionic combination might be prevented or reversed by competition from suitably charged ionic sites on proteins or lipids, or other molecules, in the environment. If the hydrophobic parts of the surfaceactive agent had a specific affinity for nonpolar structures on the bacterial surface they would become anchored at these sites and thereby determine whether the interaction with the bacterium would be permanent. In any event, a successful ionic combination must precede the second step of hydrophobic interaction. A permanent binding of the surfaceactive agent would result in irreversible damage to the bacterial surface that could initiate lysis of the bacteria. Rupture of the cellular membrane would cause leakage of soluble nitrogen and phosphorus compounds out of the cell, leading to irreparable autolysis. This process appeared to be analogous to the haemolysis of red blood cells by surface-active agents. Previously, Dresel had shown by a microscopic examination of bacteria killed by fatty acids that the fatty acids caused a complete lysis of the bacterial cells [20]. An earlier microscopical examination by Lamar [12] revealed that 0.5 to 1% sodium oleate caused a disintegration of pneumococcal cells, which turned into a mucin-like material. Pneumococci exposed to much lower concentrations of the oleate – 1 to 10 000 or 1 to 20 000 – did not show detectable changes in their morphology or in their ability to grow on culture medium. However, they underwent autolysis much more rapidly than untreated bacteria. More notably, pneumococci treated with such diluted oleate solutions

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became much more susceptible to lysis by immune antipneumococcus serum than untreated pneumococci. Lamar speculated that sodium oleate caused a weakening of the lipoidal barrier on the outer surface of the coccus, making it more pervious to the immune serum and its destructive action. Kodicek in 1949 [34] speculated about the nature of the inhibiting effect of fatty acids on bacterial growth and tried to explain it in physicochemical terms. He found former explanations, such as changed surface tension, too simplistic and reasoned that the effect was far more complex and must involve a change in the permeability of the bacterial wall. A simple calculation showed that linoleic acid in the lowest effective concentration had enough surface area to cover the whole surface of the inactivated bacteria. This supported a direct interaction of the fatty acid with the outer wall of a bacterium in which the polarity of the carboxyl group must be of importance. Certain proteins such as albumins can form complexes with fatty acids and it was well known that sterols, lecithins and other surface-active agents also formed complexes with unsaturated fatty acids. Such molecules might act as competitors to the lipoproteins of the bacterial cell wall and thus counteract the bacteriostatic or bactericidal effect of the fatty acids, as observed in many studies. Accordingly, the action of fatty acids would depend on the composition of the cell wall, as shown by the difference in susceptibility of various bacterial species to the effect of fatty acids. In spite of a physicochemical mechanism being the most likely explanation of the action of fatty acids on bacteria, Kodicek did not exclude a possible metabolic effect.

2.6

A Possible Role of Soaps and Fatty Acids in Host Defence against Bacteria

Using specific staining technics, Klotz in 1905 [43, 44] showed that calcarious tissue degeneration is preceded or accompanied by deposits of fatty or soapy material, and that such material is present in chronically inflamed tissues as neutral fats, fatty acids or soaps. Thus, prior to calcification of tissues, the cells undergo so-called fatty degeneration; that is, accumulation of fat granules which occupy the greater part of the cell. This infiltration of fat seems to play a part in the pathological process through conversion of neutral fat into fatty acids or their sodium or potassium salts, which in turn are transformed into insoluble calcium soap. Noguchi [26] made alcohol and ether extracts from various animal organs which contained mixtures of fatty acids, particularly oleic acid, and their soaps. These extracts showed haemolytic activity mostly due to their contents of oleates. The haemolytic activity of the soaps was inhibited by addition of serum. The oleate soaps were found to be highly active against B. anthracis and V. cholerae but were less active against typhoid and dysentery bacilli. In all cases, the germicidal activity of the soaps was inhibited by addition of serum. Noguchi concluded that the presence of fatty acids and soaps in blood and organs supported the opinion that they play a certain role in the natural defence mechanism of the organism. Landsteiner and Ehrlich [27] also addressed the question of the bactericidal effect of lipoid extracts from organs, particularly from leukocytes and lymphoid tissues. They confirmed a bactericidal activity of bovine spleen extracts and ovary extracts from frogs against B. anthracis, Vibrio Massauah and S. aureus and showed that serum inhibited the activity. They repeated these experiments using oleic acid emulsified in 1% saline, with or without serum added, and demonstrated bactericidal activity of the oleic acid in line with

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the organ extracts. Like Noguchi, Landsteiner and Ehrlich suggested that lipids found in organ extracts are a part of the complement system which in junction with antiserum cause lysis of bacteria and red blood cells. They concluded that further research should elucidate the possible role of lipids in the defence of the organism against infection. In the introduction to a paper by Lamar in 1911 [12], Flexner stated that recovery from bacterial infections had not been fully explained by the antibacterial activities of serum and phagocytes. Thus, he raised the question of how chemical substances, other than antibodies, might play a role in the recovery from a local bacterial infection. He quoted earlier studies by several investigators which showed that substances extracted from leucocytes are capable of killing many different types of bacteria. These intracellular substances were not only bactericidal but often also haemolytic and were characterized by their thermostability and solubility in alcohol. As mentioned above, Noguchi [26] determined in 1907 that the activity of these extracts depended on their content of certain higher unsaturated fatty acids or their alkaline soaps and Klotz [43] had shown in 1905 that soaps occur in inflammatory foci. Previously, lipase had been found in such foci, which could account for the presence of fatty acids. Disintegrating tissues were known to contain neutral fat and higher phosphorized fats, which under the influence of lipase in mononuclear leucocytes or lymphocytes were decomposed into free fatty acids. Thus Lamar [12] extracted large quantities of lipoids and fatty acids from human lung with resolving lobar pneumonia and suggested that the decrease in the number and virulence of pneumococci present in the exudate depended in part upon the presence of germicidal fatty acids and soaps. This assumption was further strengthened by the fact that pneumococci treated in vitro with soap become gradually less virulent. The quantity of bactericidal and haemolytic substances released by disintegrating cells in inflammatory foci could be considerable and was assumed to exert a destructive action upon the infecting bacteria and thus contribute to recovery from the infection. Flexner suggested that the bactericidal substances consist of soaps of fatty acids. The defensive processes acting upon bacteria in the site of infection might be complicated by the inhibiting action of serum proteins on the bactericidal action of unsaturated fatty acid soaps. It was suggested that this might be overcome by the proximity of the bacteria to the nascent fatty acids. It had been known since the beginning of the twentieth century that the human skin is able to control the microflora on its surface and to get rid of harmful bacteria coming from the environment. This ability to destroy exogenous microbes was referred to as the self-disinfecting power of the skin [45]. In the 1930s and 1940s there was an increased interest in the self-disinfecting ability of human skin and its appendages: hair, nails and cerumen. Initially, the disinfection was thought to be caused directly by the acidity of the skin but studies by Burtenshaw [46] demonstrated antibacterial activity in lipids extracted from the skin. Most of the activity was in the fractions containing oleic acid and other longchain fatty acids and their soaps. In nearly all the experiments, the disinfecting extracts and fractions thereof were far more bactericidal at an acid than at a more alkaline pH. It was concluded from these experiments that the disinfecting effect of acidity on the skin was, at least partly, due to release of antimicrobial fatty acids from their soaps at low pH. In their study of self-disinfection (self-sterilization) of the skin of young adult volunteers, Ricketts and his coworkers [47] confirmed and extended the results of Burtenshaw. Their study showed that the removal of lipids from the surface of the skin reduced its selfsterilizing power against S. pyogenes and to a lesser degree against S. aureus, but had no effect on resident micrococci or the Gram-negative B. coli and Pseudomonas pyocyanea.

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For S. pyogenes, the main disinfecting factor was the bactericidal effect of unsaturated fatty acids in the skin lipids, particularly oleic acid. On the other hand, drying of the skin was the major factor in the killing of micrococci and the Gram-negative bacilli. For S. aureus both factors seemed to have an effect. The role of antimicrobial lipids in host defence was reviewed in 2007 [48] and is discussed in detail in Chapter 6.

2.7 Studies of Prophylactic and Therapeutic Applications of Soaps and Fatty Acids Since a certain similarity seemed to exist between the destruction of pneumococci in pneumonic exudate and in artificial soap–serum mixtures, Lamar [12] speculated whether the changes in the pneumococci caused by nonbactericidal dilute sodium oleate might also occur in the animal body in the course of infection and play a role in natural defence against the infection and its final resolution. Based on these speculations, he found that a mixture of soaped pneumococci and immune serum applied by intraperitoneal inoculation was harmless to mice, in contrast to pneumococci treated with dilute oleate alone or in mixtures with normal serum. When immune serum was injected into mice within one hour after inoculation with soaped pneumococci the infection was prevented, but not if the serum was injected later. In order to ascertain whether or not a mixture of sodium oleate and immune serum could prevent infection in already infected animals, mice were inoculated with untreated virulent pneumococci and then injected with a mixture of oleate, immune serum and boric acid. The latter was needed to eliminate the inhibiting effect of serum proteins on the oleate. If the mixture was injected within one hour of inoculation it prevented the pneumococcal infection and all of the animals survived. Injection after two hours only saved half of the animals. Based on these results, Lamar speculated on whether soap–serum mixtures could be used for treatment of local pneumococcus infections in humans. Considering the possibility that Treponema pallidum, the causative agent of syphilis, could inadvertently be transmitted to small cuts in the face, for example in a barbershop, and thus cause extragenital lesions, Reasoner in 1917 [49] tested the germicidal activity of soap against this bacterium. Testicular juice from infected rabbits rich in treponemata was mixed with equal parts of a solution of various soaps prepared in distilled water. Both Ivory soap and shaving creams from various manufacturers were tested. After 15 to 30 seconds the juice was examined by dark-field microscopy. Almost no motile spirochetes could be found, and those that were found were usually swollen and distorted, whereas in control samples without soap motility could be observed for at least four hours. There was no difference in the action of the different soaps. Thus, soap solutions or lather, such as that used in shaving, immediately killed the treponemata on contact and were suggested as a possible prophylactic measure against syphilis. In a study of how germs which cause respiratory infections pass from one person to another, Nichols [14] examined the route of travel of haemolytic streptococci in an army camp. One possible point of transfer between individuals was through contaminated dishwashing water. He found that colon bacilli were on several occasions present in the water under conditions which showed no streptococci, indicating that in contrast to bacteria

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which cause intestinal disease, streptococci and pneumococci are not spread to eating utensils through this route. The bactericidal activity of the soaps was dependent on the pH and was lost if it fell to 7 or lower, probably because of precipitation of the soap from solution. He concluded that for a maximum antibacterial action, the pH of washing-up water should be kept at 8 or higher. In 1930, Renaud [50] published a paper on the possible therapeutic application of soaps. In this study he favourably used a 2% solution of sodium oleate for rinsing and as a wet dressing for wounds and abscesses, such as genital sores and tuberculous and pyogenic abscesses. Following his studies of the germicidal action of sodium salts of 18-carbon unsaturated fatty acids on Gram-positive bacteria, Hettche in 1934 [21] speculated about a possible therapeutic application for the highly bactericidal linolenate and suggested a possible use as throat wash in diphtheria or for cleaning infected wounds. In his opinion, a parental and particularly an intravenous application would not be possible because of the haemolytic activity of the lipids, whereas oral intake might be considered because of experimental evidence that oleic acid was well tolerated by mice. Chaulmoogra oil, made from the seeds of a tree common in Southeast Asia, was used for centuries to treat leprosy. Due to the reputation of this oil in the treatment of leprosy, Walker and Sweeney [51] carried out a series of experiments to test its activity in vitro against a number of acid-fast bacteria, such as the bacilli of rat and human leprosy and the bacilli of human, avian and bovine tuberculosis.The activities of the sodium salts of the total fatty acids of chaulmoogra oil were first investigated against the bacillus of rat leprosy and found to be completely antiseptic (bacteriostatic) up to a dilution of 1 : 125 000 and fully bactericidal up to a dilution of 1 : 100 000. The antiseptic and bactericidal actions of the total sodium salts were then tested against the human leprosy bacillus and the three tubercle bacilli, with results comparable to those for the rat leprosy bacillus. Notably, the bactericidal activity of sodium chaulmoograte against acid-fast bacteria was about 100-fold greater than that of the nonspecific antiseptic compound phenol. In order to identify the active ingredients of chaulmoorga oil, chemically distinct fatty acids were isolated and found to consist mostly of the cyclic 18- and 16-carbon fatty acids chaulmoogric and hydnocarpic acids. Their sodium salts were tested against the rat leprosy bacillus and found to account for the antiseptic activity of the total fatty acids, whereas palmitic acid, which constitutes only about 10% of the total fatty acids compared with 90% for the chaulmoogric acids series, had very little activity. While chaulmoogric acid was highly active against acid-fast bacteria it was inactive against eight different nonacid-fast bacteria tested, even in dilutions as low as 1 : 1000. Also, in contrast to the findings of Rogers [52], Walker and Sweeney found the sodium salt of the noncyclic 18-carbon linoleic acid as well as sodium salts of the total fatty acids of cod-liver oil, sodium morrhuate, to be much less active than sodium chaulmoogrates against leprosy and tubercle bacilli. The authors concluded that their results provided a scientific basis for the use of chaulmoogra oil and its products in the treatment of leprosy and furnished grounds for their application in the therapy of tuberculosis. Walker and Sweeney speculated about the nature of the specific action of chaulmoogric acid against acid-fast bacteria and found it unlikely that it was due to a direct effect on their protective fatty capsule, manifest by irregular acid-fast staining, as suggested by Rogers [52]. The authors hypothesized that the bactericidal action was due to an aberration in lipid

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metabolism, in which chaulmoogric acid molecules accumulated in the fatty capsules and exerted a toxic effect on the bacterial cell as a consequence of their cyclic structure. Based on an apparently successful treatment of leprosy by intravenous injection of chaulmoogra oil, Lindenberg and Pestana in 1921 [53] decided to test the activity of the oil, and fatty acids extracted from it, against leprosy bacilli, in order to establish whether or not the oil had a specific action against the causative agent. Since a culture technic for leprosy bacillus was not available to them at that time, they used cultures of human and avian tubercle bacilli and other acid-fast bacilli in their experiments. High dilutions of fatty acids from chaulmoogra oil were found to be active in preventing the growth of tubercle bacilli and other acid-fast bacteria, whereas they had much less or no effect on antrax bacilli and S. aureus. Cottonseed oil, linseed oil and cod-liver oil were also tested against tubercle bacilli. All three were found to be almost as active as chaulmoogra oil in preventing growth of the bacilli. This was considered of interest since cod-liver oil had for a long time been used as a remedy for tuberculosis. An effort was made to determine the chemical nature of the active ingredients of the oils and they were found to be mostly unsaturated fatty acids. Attempts to treat experimental animals and patients with tuberculosis with fatty acids from chaulmoogra oil, cod-liver oil and cottonseed oil by intravenous or subcutaneous injection were not successful. As reviewed by Stanley and his coworkers [54], chaulmoogra oil was used for centuries in the treatment of leprosy. Rubbing of lesions with the oil or taking it by mouth seemed to give some relief to lepers, but the latter caused side effects such as nausea. Subcutaneous or intramuscular injection of the oil was somewhat more effective than oral administration, but had the disadvantage of causing severe pain. Treatment with ethyl esters or sodium salts of the fatty acids of chaulmoogra oil was more successful, so that a small percentage of advanced cases and a much larger percentage of incipient leprosy cases became negative for the bacteria and could be discharged as cured. Stanley and coworkers studied the chemistry of the active principles of chaulmoogra oil, the 18- and 16-carbon cyclopentenyl fatty acids, chaulmoogric and hydnocarpic acids, and confirmed that neither the double bond of the ring structure nor the carboxyl group were essential for the bactericidal activity against Mycobacterium leprae. The analogous straight-chained fatty acids had no activity against the bacterium, so it was concluded that the ring structure of the molecules and the number of carbon atoms were largely responsible for the bactericidal action. Efforts by these authors to synthesize derivatives of chaulmoogric acids with higher in vitro activities against acid-fast bacteria, with the aim of improving the treatment of leprosy, were, however, unsuccessful. The use of sulfones to treat leprosy in the 1940s put an end to experiments with chaulmoogra-oil treatments. In the late 1940s, on the other hand, H¨anel and Piller [55] reported clinical improvement in patients with pulmonary tuberculosis treated by mouth with an emulsion of unsaturated fatty acids from cod-liver oil or with pharmaceutical preparations, which had been shown to be highly active in preventing growth of the tubercle bacillus in vitro. These clinical trials suggested that treatment of patients with concentrated emulsions of unsaturated fatty acids from fish oil might be beneficial. The authors reasoned that unsaturated fatty acids, in conjunction with phosphatides such as lecithin, might play a role in the natural defence of the body against the tubercle bacillus and that therapy with high doses of the fatty acids would therefore be helpful. In this context it is of interest that native populations in tropical areas have used fats against mycobacterial infections, especially leprosy and tuberculosis.

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In Nigeria palmkernel oil has been used, while turtle oils have been used by Mexican Indians. Turtle oils are rich in 10-, 12- and 14-carbon saturated fatty acids, which have been shown to be strongly bacteriostatic for Mycobacterium tuberculosis and other mycobacteria in vitro [32].

2.8

Conclusions

One hundred years ago there were few resources to fight bacterial infections. Therefore, the discovery that common soaps kill many types of pathogenic bacteria raised the hopes of health professionals and researchers that soaps might be used as disinfectants or even for prevention and treatment of infectious diseases. For more than half a century the germicidal properties of potassium and sodium salts of fatty acids were therefore actively studied. Although the results of these studies may have helped to improve sanitary practices, they did not contribute much to treatment of infections or infectious diseases. The advent of antibiotics around the middle of the twentieth century led to a breakthrough in the therapy and cure of many previously fatal infectious diseases and consequently interest in the germicidal action of fatty acids and other lipids decreased. After 1960, there was an increase in studies of antimicrobial lipids, particularly milk lipids, largely due to the pioneering work of Kabara and his associates and of Welsh and May on antiviral lipids in human milk. These studies will be described in later chapters.

References 1. Koch, R. (1881) Ueber Desinfection. Mittheil. des kaiserl. Gesundheitsamtes, 1, 234–282. 2. Reithoffer, R. (1896) Ueber die Seifen als Desinfektionsmittel. Arch. f. Hyg., 27, 350–364. 3. Serafini, A. (1898) Beitrag zum experimentellen Studium der Desinfectionsf¨ahigkeit gew¨ohnlicher Waschseifen. Arch. f. Hyg., 33, 369–398. ¨ 4. Konr´adi, D. (1902) Uber die baktericide Wirkung der Seifen. Arch. f. Hyg., 44, 101–112. 5. Behring, E.A. (1890) Ueber Desinfection, Desinfectionsmittel und Desinfectionsmethoden. Zeitschr. f. Hyg., 9, 395–478. 6. Jolles, M. (1893) Ueber die Desinfectionsf¨ahigkeit von Seifenl¨osungen gegen Cholerakeime. Zeitschr. f. Hyg., 15, 460–473. 7. Jolles, M. (1895) Weitere Untersuchungen u¨ ber die Desinfectionsf¨ahigkeit von Seifenl¨osungen. Zeitschr. f. Hyg., 19, 130–138. 8. Nijland, A.H. (1893) Ueber das Abt¨odten von Cholerabacillen in Wasser. Arch. f. Hyg., 18, 335–372. 9. Beyer, T. (1896) Ueber W¨aschedesinfection mit dreiprocentiger Schmierseifenl¨osungen und mit Kalkwasser. Zeitschr. f. Hyg., 22, 228–262. 10. Reichenbach, H. (1908) Die desinfizierenden Bestandteile der Seifen. Zeitschr. f. Hyg., 59, 296–316. 11. Geppert, J. (1889) Zur Lehre von den Antisepticis. Eine Experimentaluntersuchung. Berl. klin. Wochenschr., 26, 789–794, 819–821. 12. Lamar, R.V. (1911) Chemo-immunological studies on localized infections. First paper. Action on the pneumococcus and its experimental infections of combined sodium oleate and antipneumococcus serum. J. Exp. Med., 13, 1–23. 13. Lamar, R.V. (1911) Chemo-immunological studies on localized infections. Second paper. Lysis of the pneumococcus and hemolysis by certain fatty acids and their alkali soaps. J. Exp. Med., 13, 380–386. 14. Nichols, H.J. (1919–1920) Bacteriologic data on the epidemiology of respiratory diseases in the army. J. Lab. Clin. Med., 5, 502–511.

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15. Walker, J.E. (1924) The germicidal properties of chemically pure soaps. J. Infect. Dis., 35, 557–566. 16. Walker, J.E. (1925) The germicidal properties of soap. J. Infect. Dis., 37, 181–192. 17. Walker, J.E. (1926) The germicidal properties of soap. J. Infect. Dis., 38, 127–130. 18. Walker, J.A. (1931) The germicidal and therapeutic applications of soaps. J. Am. Med. Assoc., 97, 19–20. 19. Eggerth, A.H. (1926) The effect of the pH on the germicidal action of soaps. J. Gen. Physiol., 10, 147–160. 20. Dresel, E.G. (1926) Bakteriolyse durch Fetts¨auren und deren Abk¨ommlinge. Zentralbl. f. Bakt. I. Orig., 97, 178–181. 21. Hettche, H.O. (1934) Die Wirkung von Fetts¨auresalzen auf Erythrozyten und Bakterien. Zeitschr. Immunit¨atsforsch., 83, 506–511. 22. Barnes, L.A. and Clarke, C.M. (1934) The pneumococcicidal powers of sodium oleate and sodium ricinoleate. J. Bacteriol., 27, 107–108. 23. Bayliss, M. (1936) Effect of the chemical constitution of soaps upon their germicidal properties. J. Bacteriol., 31, 489–504. 24. Cowles, P.B. (1941) The germicidal action of the hydrogen ion and of the lower fatty acids. Yale J. Biol. Med., 13, 571–578. 25. Weitzel, G. (1952) Antituberkul¨are Wirkung unges¨attigter Fettstoffe in vitro. Zeitschr. physiol. Chem., 290, 252–266. ¨ 26. Noguchi, H. (1907) Uber gewisse chemische Komplementsubstanzen. Biochem. Zeitschr., 6, 327–357. 27. Landsteiner, K. und Ehrlich, H. (1908) Ueber bakterizide Wirkungen von Lipoiden und ihre Beziehung zur Komplementwirkung. Centralbl. f. Bakt., 45, 247–257. 28. Eggerth, A.H. (1927) The effect of serum upon the germicidal action of soaps. J. Exp. Med., 46, 671–688. 29. Dubos, R.J. (1947) The effect of lipids and serum albumin on bacterial growth. J. Exp. Med., 85, 9–22. 30. Davis, B.D. and Dubos, R.J. (1947) The binding of fatty acids by serum albumin, a protective growth factor in bacteriological media. J. Exp. Med., 86, 215–228. 31. Avery, O.T. (1918) A selective medium for B. influenzae. Oleate-hemoglobin agar. Am. Med. Assoc., 71, 2050–2051. 32. Nieman, C. (1954) Influence of trace amounts of fatty acids on the growth of microorganisms. Bacteriol. Rev., 18, 147–163. 33. Kodicek, E. and Worden, A.N. (1945) The effect of unsaturated fatty acids on Lactobacillus helveticus and other Gram-positive micro-organisms. Biochem. J., 39, 78–85. 34. Kodicek, E. (1949) The effect of unsaturated fatty acids on Gram-positive bacteria. Soc. Exp. Biol. Symp., 3, 217–232. 35. Humfeld, H. (1947) Antibiotic activity of the fatty-acid-like constituents of wheat bran. J. Bacteriol., 54, 513–517. 36. Foster, J.W. and Wynne, E.S. (1948) Physiological studies on spore germination, with special reference to Clostridium botulinum. IV. Inhibition of germination by unsaturated C18 fatty acids. J. Bacteriol., 55, 495–501. 37. Feeney, R.E., Mueller, J.H. and Miller, P.A. (1943) Growth requirements of Clostridium tetani. II. Factors exhausted by growth of the organism. J. Bacteriol., 46, 559–562. 38. Hardwick, W.A., Guirard, B. and Foster, J.W. (1951) Antisporulation factors in complex organic media. II. Saturated fatty acids as antisporulation factors. J. Bacteriol., 61, 145– 151. 39. Eggerth, A.H. (1929) The germicidal and hemolytic action of ␣-brom soaps. J. Exp. Med., 49, 53–62. 40. Eggerth, A.H. (1929) The germicidal action of hydroxy soaps. J. Exp. Med., 50, 299–313. 41. Eggerth, A.H. (1931) The germicidal action of ␣-mercapto and ␣-disulfo soaps. J. Exp. Med., 53, 27–36. 42. Hotchkiss, R.D. (1946) The nature of the bactericidal action of surface active agents. Ann. NY Acad. Sci., 46, 479–493

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43. Klotz, O. (1905) Studies upon calcareous degeneration. I. The process of pathological calcification. J. Exp. Med., 7, 633–675. 44. Klotz, O. (1906) Studies upon calcareous degeneration. II. The staining of fatty acids and soaps in the tissues by Fischler’s method, and a modification of the same. J. Exp. Med., 8, 322–336. 45. Arnold, L., Gustafson, C.J., Hull, T.G. et al. (1930) The self-disinfecting power of the skin as a defense against microbic invasion. Am. J. Epidemiol., 11, 345–361. 46. Burtenshaw, J.M.L. (1942) The mechanism of self-disinfection of the human skin and its appendages. J. Hyg., 42, 184–210. 47. Ricketts, C.R., Squire, J.R. and Topley, E. (1951) Human skin lipids with particular reference to the self-sterilising power of the skin. Clin. Sci., 10, 89–111. 48. Thormar, H. and Hilmarsson, H. (2007) The role of microbicidal lipids in host defense against pathogens and their potential as therapeutic agents. Chem. Phys. Lip., 150, 1–11. 49. Reasoner, M.A. (1917) The effect of soap on Treponema pallidum. J. Am. Med. Assoc., 68, 973–974. 50. Renaud, M. (1930) Sur le traitement des plaies et surfaces ulc´eres par les savon. Bull. et m´em. Soc. m´ed. d. hop. de Paris, 46, 1584–1589. 51. Walker, E.L. and Sweeney, M.A. (1920) The chemotherapeutics of the chaulmoogric acid series and other fatty acids in leprosy and tuberculosis. I. Bactericidal action, active principle, specificity. J. Infect. Dis., 26, 238–264. 52. Rogers, L. (1919) A note on sodium morrhuate in tuberculosis. Brit. Med. J., 1 (3032), 147–148. 53. Lindenberg, A. und Pestana, B.R. (1921) Chemotherapeutische Versuche mit Fetten an Kulturen s¨aurefester Bacillen. Zeitschr. f. Immunit¨atsf ., 32, 66–86. 54. Stanley, W.M., Coleman, G.H., Greer, C.M., Sacks, J. and Adams, R. (1932) Bacteriological action of certain synthetic organic acids toward Mycobacterium leprae and other acid-fast bacteria. J. Pharm. Exp. Therap., 45, 121–161. 55. H¨anel, F. und Piller, S. (1950) Unges¨attigte Fetts¨auren in der Therapie der Lungentuberkulose. Beitr. Klin. Tuberk., 103, 239–245.

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3 Antibacterial, Antiviral and Antifungal Activities of Lipids Gudmundur Bergsson1 , Hilmar Hilmarsson2 and Halldor Thormar2 1

3.1 3.2

3.3

3.4

3.5

Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland 2 Faculty of Life and Environmental Sciences, University of Iceland, Reykjavik, Iceland

Introduction Antibacterial Activities of Fatty Acids and Monoglycerides 3.2.1 Activities against Gram-Positive Bacteria In Vitro 3.2.2 Activities against Gram-Negative Bacteria In Vitro 3.2.3 Antibacterial Mechanism 3.2.4 A Possible Role of Antimicrobial Lipids in Host Defence against Bacteria Antiviral Activities of Fatty Alcohols, Fatty Acids and Monoglycerides 3.3.1 Early Studies of Antiviral Lipids 3.3.2 Antiviral Activities of Milk Lipids 3.3.3 Antiviral Activities of Lipids and Fatty Alcohols 3.3.4 Antiviral Mechanism of Lipids Antifungal Activities of Fatty Acids and Monoglycerides 3.4.1 In Vitro Studies 3.4.2 In Vivo Studies Conclusions

Lipids and Essential Oils as Antimicrobial Agents
C 2011 John Wiley & Sons, Ltd

Halldor Thormar

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3.1 Introduction Antimicrobial lipids are widespread in nature, where they are found in species that range from plants to animals and act as both potent antiinflammatory and antimicrobial effector molecules [1–8]. Simple natural lipids, such as fatty acids and monoglycerides, exhibit antimicrobial properties and are capable of killing Gram-negative and Gram-positive bacteria, enveloped viruses, yeast, fungi and parasites that infect both the skin and the mucosa of animals. Fatty acids and/or 1-monoglycerides are therefore considered important as target molecules for drug applications, such as antimicrobial formulations [9–11], alternatives or adjuncts to antibiotics against antibiotic-resistant bacteria [8, 12–14], emulsions [11] and potent effector molecules of innate immunity furthering bacterial clearance after skin infections [15]. The antimicrobial in vitro effect of lipids has been studied for over a century [1, 16] and although most of the early studies focussed on the bactericidal effects of soaps, longand medium-chain fatty acids and monoglycerides were also shown to kill microbes. Fatty acids and 1-monoglycerides have been found to kill pathogens that are known to infect mucosa and skin, where they are considered to be potent inhibitors of human pathogens. The main subject of this chapter is to review the in vitro antimicrobial activities of simple lipids – that is, straight-chain fatty acids, fatty alcohols and glycerides – with special focus on the comparison of the antimicrobial effects of fatty acids and their corresponding 1-monoglycerides on various groups of microbes. An overview of the most common lipids tested for antimicrobial activities is given in Table 3.1. Furthermore, possible mechanisms behind the antimicrobial effect of these lipids will be described and in vivo studies of their possible role in host defence against bacterial and fungal infections will be discussed.

3.2 Antibacterial Activities of Fatty Acids and Monoglycerides 3.2.1

Activities against Gram-Positive Bacteria In Vitro

Kabara and coworkers [17, 18] determined the minimal inhibitory concentrations (MIC) of medium-chain fatty acids against a number of Gram-positive and Gram-negative bacteria at 35 ◦ C for 18 hours. Of all the saturated fatty acids that ranged from 6 to 18 carbons in chain length, lauric acid was found to exhibit the greatest antibacterial effect against the Gram-positive bacteria that were tested. The most susceptible were pneumococci and group A streptococci, while Staphylococcus aureus, Staphylococcus epidermidis and group D streptococci were the least susceptible. Similar results were reported by Heczko et al. [19], who determined the minimal inhibitory concentrations of capric (10 : 0), lauric (12 : 0), linoleic (18 : 2) and linolenic (18 : 3) acids for 242 strains of S. aureus and 117 strains of streptococci of groups A, B, C and G. S. aureus proved to be generally less susceptible to all four fatty acids than the streptococcal strains and lauric acid was the most inhibitory fatty acid against the bacteria. To determine which lipids kill Gram-positive bacteria the fastest, Bergsson et al. investigated short incubation times of 1 to 10 minutes [20]. The results are summarized in Table 3.2, which represents the concentrations of fatty acids and monoglycerides that cause more than 100-fold (greater than 99%) reduction of viable bacteria after incubation for 10 minutes or less. The Gram-positive streptococci, group A (GAS) and group B (GBS), and

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Table 3.1 Antimicrobial fatty acids, monoglycerides and fatty alcohols. Fatty acids

Monoglycerides

Fatty alcohols

Carbon atoms : double bonds

Formic Acetic Propionic Butyric Valeric Caproic Caprylic Pelargonic/Nonanoic Capric Undecanoic Undecylenic/Undecylic Lauric Myristic Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Arachidonic

— — — — — Monocaproin Monocaprylin — Monocaprin — — Monolaurin Monomyristin Monopalmitin Monopalmitolein Monostearin Monoolein — — —

— — — — — — — — Decanol/n-decyl — — Dodecanol/lauryl Tetradecanol — — — — — — —

1:0 2:0 3:0 4:0 5:0 6:0 8:0 9:0 10 : 0 11 : 0 11 : 1 12 : 0 14 : 0 16 : 0 16 : 1 18 : 0 18 : 1 18 : 2 18 : 3 20 : 4

S. aureus were incubated with selected fatty acids at 37 ◦ C for 10 minutes. GAS and GBS were killed by 5 mM lauric acid and palmitoleic acid (16 : 1). When compared at lower concentrations, palmitoleic acid and monocaprin (10 : 0) showed considerable activities at 0.63 mM. Other fatty acids and monoglycerides had small or negligible effects. The lipids showed less activity and somewhat different antibacterial profile against S. aureus, which was susceptible to 10 mM capric acid. Similar effect has been demonstrated against Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis after as little as 1 minute incubation in milk supplemented with high concentrations (100 mM) of caprylic acid (8 : 0) [13]. It was established that antibacterial activities of long-chain fatty acids are increased by addition of a double bond [17, 18]. Addition of a second double bond to oleic acid (18 : 1) further increased the antibacterial activity but a third double bond decreased the activity. Linolenic acid has been found to exhibit strong antibacterial activity against the Gram-positive bacteria Bacillus cereus and S. aureus, an activity which can be enhanced by emulsifying agents such as monolaurin(12 : 0) and monomyristin (14 : 0) [21]. Knapp and Melly [22] investigated the antibacterial activity of polyunsaturated fatty acids and found that Gram-positive bacteria, such as Lactobacillus acidophilus and S. epidermidis, were inactivated by 0.01 mM of the essential fatty acid (EFA) arachidonic acid (20 : 4) after incubation at 37 ◦ C for one hour. More recently, several other Gram-positive bacteria have been found susceptible to low concentrations of the medium-chain lauric acid, including Propionibacterium acnes (MIC 20 ␮M) [8] and Bacillus anthracis Sterne (MIC 228 ␮M) [14].

[20] [20] [20]

Reference

a Lowest concentration tested. Highest concentration tested was 10 mM. Number of carbon atoms : Number of double bonds. Salmonella spp. and E. coli were tested for 30 minutes. NA, not active (less than 100-fold reduction); ND, not done.

NA 1.25 NA NA NA NA [26]

NA NA NA NA NA NA

NA NA NA NA NA NA

[26]

NA NA NA NA NA NA

NA NA NA NA NA NA

[26]

NA 0.31 0.15a 10a 5a NA

10 2.5 0.63 2.5 0.31a 10a

[29]

NA 0.63 0.31 NA NA NA

NA 2.5a 0.63 NA 0.63 NA

[30]

NA 2.5a NA NA NA NA

NA 10 2.5a NA NA NA

[11]

NA 10a NA NA NA NA

NA 10a NA NA ND NA

[102]

NA 10a NA NA NA NA

NA 10 5 NA NA NA

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NA 0.63 NA NA NA NA

NA 0.63 5a NA 5a NA

NA 10 10 NA NA NA

1-monglycerides Monocaprylin (8 : 0) Monocaprin (10 : 0) Monolaurin (12 : 0) Monomyristin (14 : 0) Monopalmitolein (16 : 1) Monoolein (18 : 1)

NA NA 5 NA 0.63 5a

NA NA 5 NA 0.63 5a

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Fatty acids Caprylic acid (8 : 0) Capric acid (10 : 0) Lauric acid (12 : 0) Myristic acid (14 : 0) Palmitoleic acid (16 : 1) Oleic acid (18 : 1)

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Table 3.2 Concentrations (mM) causing more than 100-fold (>99%) reduction of colony or inclusion-forming units at physiological pH in 10 minutes or less.

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The antibacterial activities of mono-, di- and triglycerides of fatty acids against a variety of bacteria have been thoroughly studied by Conley and Kabara [18] and Kabara and coworkers [17]. The spectrum of activity of monoglycerides was found to be narrower as compared to that of free fatty acids. The authors determined the MIC for glycerides against a number of Gram-positive bacteria: S. aureus, S. epidermidis and Streptococcus pyogenes. Of the monoglycerides tested, monolaurin was the most active; it was slightly more active than monocaprin against most of the bacteria and more active than the free acid. S. pyogenes was the most susceptible bacterium, particularly to monocaprin, monolaurin and monomyristin. Monolaurin and to a lesser degree monocaprin were the only monoglycerides that showed activity against S. aureus and were more active than capric acid. In addition, monolaurin was shown to kill planktonic and sessile Listeria monocytogenes cells with higher efficiency than exponential cells [23]. A comparison of the bactericidal effect of 1-monoglycerides against Gram-positive cocci after shorter incubation times [20] is given in Table 3.2. The results revealed an equal killing effect of palmitoleic acid and monocaprin against group A and group B streptococci. By using longer incubation times, lower concentrations of monocaprin were needed to kill group B streptococcus and a complete killing of the bacteria was observed after seven-hour incubation at 0.63 mM. By further testing the bactericidal effect against S. aureus [20], monocaprin proved to be the most effective of all lipids tested, causing more than 99% killing at 1.25 mM concentration. This is a similar activity profile to that observed when 1-monoglycerides were added to human milk or milk formulas [24]. 3.2.2

Activities against Gram-Negative Bacteria In Vitro

Marounek et al. studied the antimicrobial effect of fatty acids ranging from 2 to 18 carbons in chain length against Escherichia coli [25]. It was demonstrated that when the pH was dropped from 6.5 to 5.2, the medium-chain fatty acids caprylic acid and capric acid exhibited considerable inhibition and a reduction in the number of viable E. coli cells. In contrast, fatty acids ranging in chain length from 2 to 6 or from 12 to 18 carbons had negligible effect. This effect was confirmed by Nair et al., who found that 25 to 100 mM caprylic acid added to milk reduced E. coli populations [13]. As presented in Table 3.2, no fatty acids were found to exhibit bactericidal activity against E. coli or Salmonella spp. when tested at low concentrations and short incubation times for up to 30 minutes at physiological pH [26]. In contrast, the number of colony-forming units of the Gram-negative pathogen Campylobacter jejuni, which is another foodborne pathogen, was effectively reduced by 10 mM capric acid [11]. By measuring minimum inhibitory concentrations, Skrivanova et al. [27] demonstrated inhibitory effect by 21 mM caprylic acid against both E. coli and Salmonella sp. and by 29 mM capric acid against E. coli. Petschow et al. [28] found that lauric acid was the only medium-chain fatty acid that exhibited antibacterial activity against Helicobacter pylori after incubation at 1 mM for one hour. The susceptibility of H. pylori to medium-chain saturated and long-chain unsaturated fatty acids was also studied by Bergsson et al. [26]. They found 5 mM capric, lauric, myristic and palmitoleic acids caused more than one million-fold reduction in the number of viable bacteria after 10-minute incubation at 37 ◦ C. They also found 10 mM caprylic acid showed high activity against H. pylori that varied among bacterial strains. Lauric acid at a concentration of 1.25 mM and palmitoleic acid at 0.625 mM had a significant antimicrobial effect against H. pylori after 10-minute incubation. Another Gram-negative bacterium,

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Neisseria gonorrhoeae, was found to be killed by capric, lauric and palmitoleic acids [29]. Further analysis by exposure for one minute to a 2.5 mM concentration identified lauric and palmitoleic acids as the most effective fatty acids against N. gonorrhoeae [29]. Similar studies found 10 mM capric and lauric acids to cause more than 10 000-fold reduction in the number of Chlamydia trachomatis inclusion forming units in 10 minutes [30]. When tested at 5 mM concentration for five minutes, lauric acid was the most active lipid, causing more than 100 000-fold inactivation of C. trachomatis. The effect was followed by capric acid and monocaprin, but caprylic, myristic, palmitoleic and oleic acids had negligible effects against C. trachomatis [30]. The long-chain polyunsaturated arachidonic acid (20 : 4) also killed the Gram-negative bacteria Neisseria and Haemophilus spp. In contrast, E. coli was not susceptible to arachidonic acid, even after six-hour incubation with 0.3 mM arachidonate [22]. As to studies on the effect of glycerides of fatty acids on Gram-negative bacteria, Conley and Kabara [18] and Kabara et al. [17] found that di- and triglycerides did not exhibit antibacterial effects and none of the compounds tested inactivated E. coli or a number of other Gram-negative bacteria. Further studies by Isaacs et al. [24] showed that when medium-chain saturated monoglycerides were added to human milk and milk formulas the mixtures inactivated Haemophilus influenzae. The same was not true for polyunsaturated long-chain monoglycerides, which were less active. Petschow et al. [28] also studied the antibacterial effect of medium-chain monoglycerides against H. pylori and found that incubation of the bacteria with 1 mM monoglycerides of 10-, 12- and 14-carbon fatty acids for one hour at 37 ◦ C caused a 10 000-fold reduction in the number of viable bacteria. Lower activities were observed after incubation with monoglycerides with shorter- or longer-chain fatty acids, that is 9-, 15- and 16-carbon chains [28]. The antibacterial effect of 1-monoglycerides against Gram-negative bacteria was also tested at incubation times of 1 to 10 minutes by Bergsson et al. [26, 29, 30], as shown in Table 3.1. Monocaprin, monolaurin and monopalmitolein (16 : 1) at 5 mM concentration were all highly effective in killing H. pylori in 10 minutes. At lower concentrations (0.625 mM) and after one minute incubation time, monocaprin and monolaurin were the only lipids showing significant activities against H. pylori [26]. The Gram-negative bacterium N. gonorrhoeae was found to be killed by 0.31 mM monocaprin and 0.63 mM monolaurin in 10 minutes [29]. All other monoglycerides showed much smaller effect. The Gram-negative pathogens H. pylori and N. gonorrhoeae were found to be exceptionally susceptible to lipids, both fatty acids and 1-monoglycerides, when compared to other bacteria studied, either Gram-negative or Gram-positive. The effects of 1-monoglycerides were also studied against C. trachomatis [30]. The studies found monocaprin to be the most effective glyceride, causing more than 100 000-fold reduction in viable bacteria after five minutes at 5 mM concentration. A 50% effective concentration of 0.12 mM was found by incubation of C. trachomatis with monocaprin for two hours. C. trachomatis was not susceptible to 10 mM monocaprylin, monolaurin, monopalmitolein or monoolein (18 : 1). A study of the susceptibility of Campylobacter jejuni to a series of monoglycerides found monocaprin to be particularly active in killing this bacterium [11]. In contrast to the above Gram-negative bacteria, E. coli and Salmonella spp. were found to be resistant to all monoglycerides tested at neutral pH [26]. However, in an acidified buffer at pH < 5, monocaprin was highly effective in killing both E. coli and Salmonella, reducing the number of colony-forming units by more than one million-fold in 10 minutes

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[11]. Furthermore, Nair et al. [13, 31] showed that when monocaprylin was added in high concentrations (50 mM) to milk or at 5 mM to apple juice, it effectively killed E. coli. On the other hand, Preuss et al. [14], in a study of the bactericidal effect of essential oils and monolaurin on bacteria, confirmed that E. coli and Klebsiella pneumoniae were very resistant to this monoglyceride, whereas H. pylori was susceptible. In general, the bactericidal activities of fatty acids and monoglycerides produced by different laboratories are in agreement. Any discrepancies may be due to differences in experimental conditions, particularly differences in lipid concentrations and incubation times. Gram-positive and many Gram-negative bacteria have been found to be highly susceptible, with the exception of Enterobacteriacae, which in most studies have proved to be resistant at neutral pH. 3.2.3

Antibacterial Mechanism

Kabara [1, 16] made generalizations concerning the relationship between lipid structure and antibacterial activity. He concluded that 1) lauric acid is the most active saturated fatty acid, palmitoleic acid the most active monounsaturated acid, and linoleic acid the most active polyunsaturated fatty acid against Gram-positive bacteria, 2) monoglycerides of medium-chain saturated fatty acids are more active than the free acids, particularly the monoglyceride of lauric acid, 3) fatty acids have very low activity against Gram-negative bacteria except when having a very short chain (6 carbons or less), and 4) yeasts are affected by fatty acids with short chain lengths (10 to 12 carbons). The most important generalization is probably that monoesters of fatty acids, such as monolaurin, are very active, while di- and triglycerides are not. From these studies it was concluded that the antibacterial effect of short-chain (6 to 10 carbons) saturated fatty acids is related to the degree of their dissociation, and they were found to be more active at pH 6.5 than at pH 7.5. These early studies established that unsaturated fatty acids are active against Gram-positive bacteria and indicated that they become more active with an increasing number of double bonds [1, 16]. Further studies by Kabara and coworkers demonstrated that the activities of long-chain fatty acids were increased by addition of a double bond. Addition of a second double bond to oleic acid (18 : 1) further increased the antibacterial activity, but a third double bond decreased the activity [17, 18]. The ability of lipids to disrupt cellular membranes has been demonstrated for bacterial cells, both Gram-negative and Gram-positive. As mentioned above, monocaprin and monolaurin had little or no activity against Gram-negative bacteria such as E. coli at 30 ◦ C [32] and physiological pH [26]. However, when E. coli was incubated with lauric acid, monocaprin and monolaurin at 50 ◦ C for five minutes, or with monocaprin at lower pH [11], a remarkable inactivation was observed, much more than that produced by heating alone. Cells growing in the logarithmic phase were more sensitive than stationary-phase cells. Taken together, this suggests a reduction in the permeability barrier in the outer membrane caused by heating, allowing penetration of the lipid through the outer membrane and the cell wall. Increased transfer of monolaurin into bacterial cells was seen when incubating E. coli in a mixture of monocaprin or monolaurin with citric acid. These studies suggest that the acid removes a barrier in the outer membrane of the cell wall, giving the lipid access to the inner membrane, or that lower pH will increase the concentration of the undissociated form of the fatty acids. In summary, saturation of natural antimicrobial lipids affects the

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lipid ability to cause disintegration of microbial membranes. This effect can be enhanced by altering environmental factors. Hypotheses concerning the antimicrobial mechanism of fatty acids and their 1monoglycerides have been tested by several authors. Complete cell disorganization and disruption were seen by Knapp and Melly in N. gonorrhoea cells after incubation with 0.05 mM arachidonic acid for one hour [22].These authors obtained similar results for H. influenzae, while no morphological changes were noted in E. coli, which is not susceptible to arachidonic acid. Peripheral cytoplasmic condensations were visualized in S. aureus and Lactobacillus acidophilus, while no changes were visible in the cell walls. As illustrated in Figure 3.1, the viability of the Gram-negative bacterium C. trachomatis was irreversibly lost after treatment with monocaprin [30]. The negatively stained elementary bodies of C. trachomatis appeared deformed and partly disintegrated after 10 minutes, suggesting that monocaprin kills the bacteria by disruption of the cellular membrane(s) (Figure 3.1) [30]. This effect is similar to that of glyceryl ethers, 1-O-hexyl-sn-glycerol [33]. The effect of monocaprin on the structure of Gram-positive cocci was studied by transmission electron microscopy (TEM) (Figure 3.2), and with a two-colour fluorescent bacterial viability kit to stain monocaprin-treated group B streptococci [20]. A treatment with 10 mM monocaprin for 10 minutes caused damage to the cell membrane, which became permeable to propidium iodide. Examination of thin sections of bacterial cells in TEM confirmed this finding (Figure 3.2), since after treatment with monocaprin the plasma membrane was no longer visible, indicating a disintegration of the membrane. No changes were detectable on the surface or in the structure of the bacterial cell wall by scanning electron microscopy (SEM). This suggests that the lipids can penetrate the cell wall of Gram-positive bacteria and cause disintegration when it reaches the bacterial cell membrane. The differences in the susceptibilities of Gram-negative bacteria to antimicrobial lipids are notable, particularly between H. pylori and Enterobacteriacae [26], and may be due to differences in the outer membrane of the bacteria. In this regard, the external leaflet of the outer membrane of E. coli and Salmonella is almost entirely composed of lipopolysaccharides (LPS) and proteins. These bacteria have a hydrophilic surface due to the O-side chains of the LPS, making it difficult for hydrophobic molecules, like lipids, to enter the bilayer. Moreover, molecules may have difficulty in penetrating these membranes because of the low fluidity of the hydrocarbon chains in the LPS leaflet and the strong interactions between the LPS molecules. On the other hand, Gram-negative bacteria with a relatively hydrophobic surface, such as N. gonorrhoeae [34], are easily killed by medium-chain fatty acids and monoglycerides [29]. The outer membrane of N. gonorrhoeae is not composed of LPS but of lipooligosaccharides (LOS) that lack the O-antigen side chain. Even if LOS is mainly expressed on mucosal bacteria, it can also be expressed on enteric bacteria such as Campylobacter spp. [34], which have been found to be susceptible to monocaprin [11]. Desbois and Smith in 2010 [35] reviewed the mechanisms of antibacterial actions of fatty acids. A variety of detrimental effects can be caused by partial solubilization of the cell membrane by fatty acids, leading to release of membrane proteins. This can result in interference with the electron transport chain and uncoupling of oxidative phosphorylation, or to inhibition of enzyme activity and nutrient uptake, causing inhibition of bacterial growth. At higher concentrations, the detergent effect of fatty acids can kill bacteria by cell lysis. Thus the action can be either bacteriostatic and reversible or bactericidal and irreversible.

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(a)

(b)

(c)

(d)

Figure 3.1 Electron micrographs of negatively stained elementary bodies (EBs) of C. trachomatis. The EBs were untreated (a) and treated with 10 mM monocaprin for 1 minute (b), 5 minutes (c) and 10 minutes (d). After treatment for 10 minutes, the EBs appear deformed and shrunken or partially disrupted (d, inset). Bars, 1 µm. (Reprinted from [30] with permission from American Society for Microbiology.)

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(b)

(c)

(d)

(e)

(f)

Figure 3.2 Effects of monocaprin on the ultrastructure of group B streptococci as demonstrated by TEM of ultrathin sections of bacterial cells. (a), (c) and (e) show cells from the control samples with intact plasma membrane (PM) and intact cytoplasmic granules (G). (b), (d) and (f) show cells from samples treated with 10 mM monocaprin for 30 minutes, demonstrating disappearance of plasma membranes and cytoplasmic granules. Some changes can be seen in the cell wall (CW). (Reprinted from [20] with permission from John Wiley and Sons, Ltd.)

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Lipids such as fatty acids have to permeate the cell wall of Gram-positive bacteria and the outer membrane of Gram-negative bacteria to gain access to the bacterial cell membrane. As discussed above for Gram-negative bacteria, differential susceptibility of bacterial species to the action of lipids is most likely due to differences in these outer structures, which can act as a barrier and protect the inner cell membrane. Exposure of strains of the Gram-positive bacterium S. aureus to long-chain unsaturated fatty acids has been shown to upregulate the expression of genes associated with peptidoglycan synthesis, thus increasing the thickness of the bacterial cell wall. An adaptive response which reduced the surface hydrophobicity of the wall was also observed, making the bacteria more resistant to the action of the fatty acids [36, 37]. 3.2.4

A Possible Role of Antimicrobial Lipids in Host Defence against Bacteria

It has been known for decades that sweat, sebum [38] and breast milk [3, 39] contain lipids which are highly microbicidal and it has been suggested that they play an important role in the innate defence mechanism against microbial infections on the surface of skin and in the stomach of suckling infants. These lipids are mostly fatty acids released from sebaceous triglycerides by lipases of the normal bacterial flora [40, 41], fatty acids synthesized by sebocytes [42–44] and keratinocytes [45], and fatty acids in the cream fraction [46] of breast milk, which becomes activated upon enzymatic digestion of triglycerides by lipases [47]. Aly et al. [48] demonstrated that S. aureus, S. pyogenes and Candida albicans diminished in number when deposited on normal human skin. In contrast, acetone-washed forearms gave greater persistence of these bacteria and when these extracts/washes were replaced on the skin, the recolonization was prevented. These authors further pointed out a correlation between in vitro studies on acetone lipid extracts from skin and the microbes localized to skin. Interestingly, S. pyogenes, which rarely colonizes skin, is extremely sensitive to skin lipids [49]. Free fatty acids of human stratum corneum, as well as both polar lipids and glycosphingolipids, exhibit antistaphylococcal activity [45] at concentrations 10 to 100 times lower than the concentrations calculated to be present in the stratum corneum intercellular domain of the skin [50]. This suggests that stratum corneum lipids are effective in physiologically relevant quantities. More recently, lipids isolated from sebum have been shown to reduce the growth of Gram-positive bacteria such as S. aureus, Streptococccus salivarius and Fusobacterium nucleatum, while most Gram-negative bacteria are resistant to sebum lipids [51]. It was shown that both saturated and unsaturated fatty acids are responsible for the antimicrobial activity and that lauric acid is the most potent of the saturated fatty acids. However, a palmitoleic acid isomer, an exclusive lipid of sebum [52], was found to account for most of the fatty acid antimicrobial activity. The antimicrobial effect of milk lipids, fatty acids and monoglycerides, has been demonstrated for a variety of both Gram-positive and Gram-negative bacteria [2, 24, 47]. It was demonstrated that fivefold-diluted stomach contents of infants fed human milk or infant formula had varying degrees of activity against both S. epidermidis and E. coli after 30 minutes at 30 ◦ C [47]. The same authors further illustrated that infant formulas treated with lipases for one hour at 37 ◦ C were active against the Gram-positive bacteria S. epidermidis but not against Gram-negative bacteria, including E. coli and Salmonella enteritidis. The discrepancies in the results on E. coli may reflect incomplete lipolysis of formula

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triglycerides in lipase-treated mixtures. Additional studies demonstrated inactivation of the Gram-negative bacterium H. influenzae by 1-monoglyceride-supplemented human milk and infant formulas after 30 minutes at 37 ◦ C. Medium-chain saturated monoglycerides showed activity but polyunsaturated and long-chain monoglycerides were less active. Lipids located to a cream-like substance that covers the skin of foetuses and newborn babies have been shown to potentiate the antimicrobial action of other effector molecules of innate immunity, namely antimicrobial peptides, in vitro [53], demonstrating the importance of the microenvironment for the function of antimicrobial components. This has been further studied in other recent studies, where enhanched synergistic effect in the antimicrobial activity of monolaurin has been documented when combined with various environmental and biological factors, such as temperature changes [32, 54, 55], organic acids [56], lower pH [25, 57], the chelating agent EDTA [58], antimicrobial peptides [53, 59] and the lactoperoxidase system [55], which also enhanced the effect of other antimicrobial lipids. The presence of natural antimicrobial lipids on skin and mucosa suggests that lipids might be useful as the active ingredients in pharmaceutical formulations for prevention and treatment of skin and mucosal infections [60]. Among the clinical symptoms suggesting the importance of fatty acids as part of the innate defence mechanism is that essential fatty acid deficiency causes increased susceptibility to infections and poor wound healing [61]. Essential fatty acids are unsaturated fatty acids of 18-, 20- or 22-carbon chain lengths that are not synthesized by the human body. Essential fatty acid deficiency was first described in 1929 by Burr and Burr, who noted that poor growth of rats and skin lesions could be prevented by linoleic acid [61]. A clear microbiological difference was observed between mice that were fed a diet lacking essential fatty acids and mice fed an ordinary diet including essential fatty acids. The mice fed a diet lacking essential fatty acids contained 100-fold higher levels of bacteria on their skin than mice fed an ordinary diet. Furthermore, the mice fed diet lacking essential fatty acids were commonly colonized by S. aureus. Surprisingly, in vitro experiments demonstrated that lipids isolated from mice fed a diet lacking essential fatty acids were more lethal to bacteria commonly found on skin than lipids isolated from mice fed a normal diet. This can possibly be explained by the results of Georgel et al. [15], who studied innate immunodeficiency in mice caused by a mutation in a fatty acid desaturase (stearoyl-CoA desaturase), which is an enzyme that is essential for synthesis of monounsaturated fatty acids in sebaceous glands. These authors demonstrated that this mutation resulted in a reduction in sebum production of palmitoleic acid and oleic acid and that it is responsible for the lack of bacterial clearance after skin infections. It was further shown that the expression of stearoyl-CoA desaturase is induced by S. aureus, a bacterium susceptible to monounsaturated fatty acids, but not by E. coli [15], which is resistant to monounsaturated fatty acids [26].

3.3 Antiviral Activities of Fatty Alcohols, Fatty Acids and Monoglycerides 3.3.1

Early Studies of Antiviral Lipids

The first reports that fatty acids and their soaps might inactivate viruses appeared in the 1930s. Begg and Aitken [62] reported that potent Rous sarcoma filtrates had been inactivated by addition of 1% solution of sodium oleate. Also, intratumoural injections of sodium oleate

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sometimes led to definite regression and occasionally to complete disappearance of Rous sarcomas in fowles. Helmer and Clowes in 1937 [63] found that an unsaturated fatty acid fraction from pig pancreatic tissue had a strong inhibiting activity against the agent of chicken sarcoma. A commercial oleic acid was shown to have a comparable activity. A number of free fatty acids were tested against the agent by addition to an aqueous extract of chicken tumour. The extract was then tested for infectivity by intradermal injection into chickens. The straight-chain 18-carbon unsaturated fatty acids had a much greater activity against the tumour agent than the saturated stearic acid. However, the number of double bonds had no effect on the activity, oleic, linoleic and linolenic acids all being equally active. The length of the carbon chain had a strong influence on the activity. The lower acids, either saturated or unsaturated, had little or no activity compared to the long-chain acids. However, the activity of the long-chain acids was limited by the lack of solubility in water. Only fatty acids and their soaps were active, whereas the glycerides found for example in olive oil, cod liver oil and sardine oil had no action. Pirie had observed in 1933 [64] that pancreatic extracts destroyed tumour viruses, but the active compounds were unknown. In a further study carried out by Pirie [65], preliminary experiments indicated that the virucidal activity was associated with a fairly unstable fatty acid. Several lipid fractions were therefore separated from dried pig pancreatic extract and tested for virucidal activity against vaccinia and Fujinami tumour virus. The lecithin and fatty acid fractions were found to account for the whole virucidal power of the dried extract and the acids were apparently responsible for most of the activity. The cephalin fraction had no effect. A number of commercially obtained fatty acids were shown to have virucidal activities. Of the higher fatty acids, only the unsaturated ones, such as oleic and linoleic acids, were virucidal. Pirie speculated that ‘the fatty acids are adsorbed on the surface of the viruses and disrupt it to such an extent that the virus is lysed.’ In 1940, Stock and Francis [66] studied the inactivation of influenza virus by various fatty acids and their soaps. A virus suspension, made from the lungs of infected mice, was mixed with a fatty acid or soap solution at pH 7.5 and allowed to stand at room temperature. After 90 minutes and again after 24 hours, samples were tested for infectivity by intranasal inoculation into mice. Preliminary experiments showed that oleic and linoleic acids inactivated the virus while myristic and undecylenic (11 : 1) acids did not. A large number of fatty acids of increasing chain lengths, both saturated and unsaturated, and other lipids were then tested in the same way. The 18-carbon unsaturated fatty acids, oleic, linoleic, linolenic and chaulmoogric acids, were the most virucidal, whereas their isomers such as elaidic acid were ineffective. Of the shorter-chain saturated acids, the 12-carbon lauric acid was the most active, but the unsaturated 11-carbon undecylenic acid was ineffective. Other lipids, such as lecithin, had no effect. The results were interpreted to show a relationship between the configuration of the fatty acid molecule and the capacity to inactivate virus. The most likely mechanism for inactivation of the virus by long-chain unsaturated fatty acids was thought to be a more or less specific adsorption of fatty acid to proteins on the viral surface. This adsorption, which would probably be dependent on the configuration of both the fatty acid and the protein molecules, might result in dispersion of the protein layer on the viral surface. Experiments in which various dilutions of virus were mixed with different concentrations of oleic acid suggested that there exists a minimum concentration of oleic acid for effectively inactivating the infectivity of a given concentration of virus [67]. Notably, inactivation of the infectivity of influenza virus

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by oleic acid did not destroy the antigenicity of the virus, since mice vaccinated with the noninfective mixtures of virus and fatty acid showed a strong immune reaction [66]. However, the reaction was not as strong as in mice receiving untreated active virus [67]. On the other hand, influenza virus which had been inactivated by oleic acid was apparently more active as an antigen than other noninfectious preparations such as virus inactivated by formalin. Inactivation of lymphocytic choriomeningitis (LCM) virus by fatty acids and soaps was also studied by Stock and Francis [68]. The experiments were carried out in a similar way to the influenza virus experiments, except that the infectivity of samples was tested by intracerebral inoculation of mice. As in the previous studies of influenza virus, the 18-carbon unsaturated fatty acids were the most effective in inactivating the LCM virus. Of the saturated fatty acids, only myristic acid was effective, whereas in contrast to influenza virus, lauric acid was ineffective. As in the studies of influenza virus, the relative amounts of virus and oleic acid were found to be important in obtaining inactivation of LCM virus. In contrast to oleate-inactivated influenza virus, LCM virus inactivated in the same way failed to induce any detectable immunity in mice. This was in line with the failure to use formalinized LCM virus as an immunizing agent. 3.3.2

Antiviral Activities of Milk Lipids

Around 1960, human milk was shown to reduce the infectivity of a number of viruses. The activity against enveloped viruses, such as herpes simplex virus type 1 (HSV-1), was different from that against poliovirus, which has no lipid envelope. The antipoliomyelytic activity was bound to the protein fraction of the milk, which had no effect against enveloped viruses, while the cream fraction showed the antiherpetic activity [69]. Sarkar et al. [70] showed that whole, skimmed or cream-fraction human milk incubated with mouse mammary tumour virus for 18 hours at 37◦ C caused decreased viral infectivity. The cream fraction was more effective in the inactivation of the virons than the whole or skimmed milk. In the following years these findings where confirmed by the same authors and others [1]. Thus, the lipid fraction of milk and colostrum was found to inactivate dengue virus, which is an enveloped virus [71]. Free fatty acids and monoglycerides in milk showed antiviral activities against Semliki Forest virus, whereas diglycerides had no activity [39]. A series of studies of the antiviral properties of human milk showed that fresh milk samples are inactive against enveloped viruses such as HSV-1, vesicular stomatitis virus (VSV), measles virus and human immunodeficiency virus type 1 (HIV-1). However, most of the milk samples became highly antiviral after storage at 4 ◦ C for a few days, reducing the virus titre 1000fold in 30 minutes at 37 ◦ C. Similar antiviral activities were found in stomach samples from suckling infants who had been fed fresh human milk or infant formula feeds one hour earlier. The antiviral activity was found in the cream fraction and it was concluded that it was due to milk lipase, particularly lipoprotein lipase, releasing fatty acids from triglycerides upon storage of human milk samples. In the stomachs of infants the activity was due to gastric and lingual lipases releasing fatty acids from milk fats [2, 3, 24, 47, 72, 73]. These findings confirmed and further extended the importance of lipases for antiviral activities of milk, which had been pointed out earlier by Welsh and coworkers [39]. A comparative study of purified fatty acids normally found in human milk and other food products showed that short-chain (4 to 8 carbon) and long-chain (16 to 18 carbon) saturated fatty acids had no or minor antiviral effects against the enveloped viruses VSV, HSV-1 and visna virus

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(VV). In contrast, medium-chain saturated (10 to 14 carbon) fatty acids inactivated all the viruses, although myristic acid caused only 1.7 log10 reduction against VV after 30 minutes at 37 ◦ C [74]. Long-chain (16 to 20 carbon) unsaturated fatty acids were also active and their activities increased with increasing number of double bonds. Thus, arachidonic acid was the most active fatty acid in lower concentrations. Corresponding 1-monoglycerides of several fatty acids were also tested and all, except monomyristin and monoolein, were 5 to 10 times more active against VSV and HSV-1 than the free fatty acids. These findings suggest that lipolysis of triglycerides from human milk, either in the gastrointestinal tract or by storage, releases two types of antiviral lipids: monoglycerides and free fatty acids [74, 75]. 3.3.3

Antiviral Activities of Lipids and Fatty Alcohols

Sands [76] studied the effect of chemically purified fatty acids on the enveloped bacteriophage
6. The results showed that unsaturated long-chain fatty acids, that is palmitoleic and oleic acids, inactivated the virus, while the saturated myristic and palmitic (16 : 0) acids were inactive against
6. The lipid-containing bacteriophages
6, PM2 and PR4 were further studied by Sands and coworkers [77] for antiviral susceptibilities to free fatty acids, fatty acid derivates, mono- and diglycerides and long-chain alcohols. Low concentrations (50 ␮g/ml) of medium-chain saturated and long-chain unsaturated alcohols caused at least a 99% inactivation of
6 after 30 minutes at 25 ◦ C. Long-chain unsaturated fatty acids and corresponding monoglycerides were also antiviral, while diglycerides caused minor or no inactivation. PM2 and PR4 bacteriophages, which lack external envelopes, were much less susceptible to inactivation by the lipids. HSV type 2 (HSV-2) was inactivated by many of the lipids that were active against
6, where monoolein and monopalmitolein were found to be the most active. Snipes and coworkers [78] studied inactivation by alcohols ranging from 4 to 18 carbons on lipid-containing viruses. Saturated alcohols with chain lengths from 10 to 14 carbons showed the most antiviral activity, suggesting that a proper balance between the polar and hydrophobic components can lead to maximal perturbation of the viral membrane. Decanol (10 : 0), dodecanol (12 : 0) and tetradecanol (14 : 0) at 0.5 mM readily inactivated HSV after 30-minute exposure, as well as the bacteriophage
6, even at lower concentrations. The lipid-containing virus PM2 was susceptible to decanol and dodecanol, while tetradecanol had neglible antiviral effects. Phytol, a naturally occurring branced-chain alcohol found in chlorophyll, was active against
6 in 0.001 mM and HSV in 1 mM concentration, but not against PM2 after 30-minute exposure. The nonenveloped polyoma virus and bacteriophage
23-1-a were not inactivated by any of the alcohols tested. At 0.05 mM concentration none of the alcohols showed cytotoxic effects on human embryonic lung cells, although several of the alcohols were active against HSV at this concentration. Saturated alcohols and n-alkyl derivates of butylated hydroxytoluene (BHT) tested against
6 and HSV-2 showed that alcohols with 10 to 14 carbons in chain length were the most effective. Also, alcohols and BHT were all more active against
6 than against HSV-2. In addition, in vivo studies with dodecanol against HSV skin lesions in hairless mice revealed effective clearence of virus lesions when compared to untreated control mice [78, 79]. Thus, these early studies established that inactivation of enveloped viruses by lipids varies greatly, depending on both the nature of the lipid and the type of virus.

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Kohn and coworkers [80] studied the incorporation of essential unsaturated fatty acids, that is oleic, linoleic and arachidonic acids, into phospolipids of animal cells, which induced a change in the fluidity of the membranes and loss of rigidity. Micromolar concentration of these fatty acids (10 to 50 ␮g/ml) applied for 15 minutes to enveloped viruses such as arbo-, myxo-, paramyxo- and herpesviruses caused a rapid loss of infectivity, as measured by plaque assay. However, the abovementioned fatty acids in concentrations up to 25 ␮g/106 cells/ml did not affect the growth of baby hamster kidney cells. No antiviral activities were found for linoleic acid when incubated against the non-enveloped poliovirus, siman virus 40 or encephalomyocarditis virus, suggesting destructive lipophilic effects of the fatty acids against enveloped viruses associated with disintegration of the virus envelope [81] Thormar et al. [75] studied the effects of free fatty acids, monoglycerides and ethers against VV, which is a lentivirus related to HIV. Ethers of 6-, 7- and 8-carbon saturated fatty acids all showed activities against the virus after 30 minutes at 37 ◦ C. The most effective ether was 1-O-octyl-sn-glycerol, which is an 8-carbon ether of caprylic acid and was much more active than caprylic acid alone or its monoglyceride ester, monocaprylin. Also 1-O-octyl-sn-glycerol was found to inactivate HIV in human plasma [82]. Hilmarsson et al. [83] studied the virucidal effects of medium- and long-chain (8 to 18 carbon) fatty alcohols and corresponding lipids against HSV-1 and HSV-2 at various concentrations, times and pH levels. After 10-minute incubation at 37 ◦ C in 10 mM concentration, 14 of the lipids tested caused a significant reduction in HSV infectivity. N-decyl (10 : 0) alcohol, capric, lauric, myristic, palmitoleic, and oleic acids, and monocaprylin, monocaprin and monolaurin monoglycerides were all fully active – that is, no virus was detectable after the treatment – causing 100 000-fold or greater reduction in HSV titre. A generally good agreement was found between the activity profiles for HSV-1 and HSV-2. At shorter incubation time, monocaprin, monolaurin and all of the fatty acids except caprylic and capric acids acted rapidly, causing a significant reduction in virus titre after only one minute at 10 mM concentration. Further comparison of 10 to 12 carbon alcohols and the corresponding lipids at lower concentrations and increased incubation times against HSV-1 showed a complex relationship between concentration and duration of action. N-decyl alcohol acted rapidly at 5 mM concentration but at lower concentrations the activity was greatly reduced, even with increased incubation time. Lauryl alcohol acted more slowly compared to n-decyl alcohol, but was active at much lower concentrations (0.625 mM), particularly with increasing time, which is in agreement with previous studies by Snipes and coworkers [78, 79]. The three most active alcohols and the corresponding fatty acids and monoglycerides were further tested at pH 7 and 4.2. Lowering the pH to 4.2 caused a more rapid inactivation of HSV-1 by n-decyl and lauryl alcohols and capric acid, with a significant reduction of virus titre in one minute. The virucidal activities of all the alcohols, fatty acids and monoglycerides tested in 10 minutes at concentrations as low as 0.15 mM for monolaurin were greatly increased at pH 4.2 as compared with pH 7. It was hypothesized that the difference between the polar groups of alcohols and fatty acids, that is hydroxyl group versus carboxyl group, and the corresponding difference in their hydrophile–lipophile balance (HLB) might explain their different activities against HSV. Capric and lauric acids are hydrophilic at pH 7 with HLB numbers around 20, whereas n-decyl and lauryl alcohols are lipophilic with HLB numbers of 4.15 and 3.2, respectively. This may possibly explain the differences in activity profiles of fatty acids and alcohols seen in this study. However, the distinct differences in activity between various fatty acids,

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for example capric and lauric acids, and between various alcohols, for example n-decyl and lauryl alcohols, can hardly be explained by the small difference in HLB. In general, no correlation could be found between virucidal activities and HLB numbers. This was most obvious when activities were compared at different pHs. Alcohols and monoglycerides were much more active at pH 4.2 than at pH 7, although their HLB numbers did not change between these pH levels. The increased virucidal activity against HSV at low pH may be due to ionic changes in the surface of the host cell-derived viral envelope, which contains virus-encoded glycoproteins, somehow giving the alcohols, fatty acids and monoglycerides better access to the lipid bilayer of the envelope. Alcohols and lipids were, in studies similiar to those of HSV-1 and 2, also tested against VV to further compare the virucidal profiles against viruses and possibly elucidate how lipids inactivate enveloped viruses [84]. In this comparative study it was shown that fatty acids were most active against VV, particularly lauric, palimitoleic and oleic acids, when tested either in a low concentration or at short incubation time. N-decyl and lauryl alcohols and monocaprin and monolaurin also had significant activities, with about 100- to 1000-fold reduction after 10 minutes in 10 mM concentration. Studies on virucidal activities at low pH against VV revealed that lipids became generally more virucidal at pH 4.2 compared with pH 7, which is in agreement with studies on HSV. Previous studies by Thormar et al. [74,75] showed that certain fatty acids and monoglycerides were active against VV, which is compatible with these results. However, the data are not fully comparable since in the earlier study the incubation time was longer, that is 30 minutes. Previous studies on other enveloped viruses, such as HSV, showed that lipids and the corresponding alcohols with 10- and 12-carbon chain-length were the most virucidal, especially monocaprin [83]. This is in contrast with the results on VV, where only fatty acids had high virucidal activites. Hilmarsson et al.[85] also studied the virucidal activities of alcohols and corresponding lipids against respiratory syncytial virus (RSV) and human parainfluenza virus type 2 (HPIV2) at different concentrations, times and pH levels. Monocaprin, which has been found to exhibit broad-spectrum and fast-acting virucidal activities against a number of enveloped viruses, was also tested against influenza A virus. The comparison of virucidal activities of fatty alcohols and lipids against RSV showed that eight of the compounds tested at 10 mM concentration for 10 minutes reduced the infectivity titres up to 4.5 log10 or greater. A good agreement was found between the activity profiles for RSV ATCC (American-type culture collection) and three recent clinical isolates of RSV. Lauric, palmitoleic and oleic acids and monocaprin all acted rapidly in 10 mM concentration, causing a significant titre reduction in only one minute against all RSV strains. The most active monoglyceride tested was monocaprin, which was still highly active after dilution to 1.25 mM. Lauric acid was the most active fatty acid and N-decyl alcohol the most active alcohol in lower concentrations tested for 10 minutes. A generally good agreement was seen in the virucidal activities of the 10- and 12-carbon compounds against HPIV2 and RSV, and also for monocaprin tested against influenza A virus in decreasing mM concentrations, which revealed about 10 000-fold reduction in influenza A virus infectivity at 1.25 mM monocaprin in 10 minutes. By changing the pH from 7 to 4.2, the activities of most of the compounds were greatly increased at both lower concentrations and shorter incubation times, which is in agreement with the previous studies on HSV and VV. Thus, lauric acid in 0.62 mM concentration was fully active against both RSV and HPIV2 at pH 4.2 after 10-minute exposure, whereas it

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had no activity at pH 7. However, there was a distinct difference in the effect of low pH on the activity of monocaprin and monolaurin against these viruses, since in contrast to RSV there was no increase in the activity of the monoglycerides against HPIV2 at pH 4.2. These findings, and the well-established difference in the envelope proteins of RSV and HPIV2, further supports the hypothesis that the increased virucidal activities of lipids at low pH are due to ionic changes in the glycoproteins on the surface of the viral envelope somehow leading to a better access to the lipid bilayer of the envelope. In an extension of this study, the most active lipids were mixed in 10 mM concentration with milk products and fruit juices and the mixtures tested for virucidal effects against RSV. N-decyl alchol, lauric acid, monocaprin and monolaurin were found to preserve their activities against RSV to varying degrees in such mixtures. The experiments revealed that all four compounds were fully active in pear juice and monolaurin and monocaprin were also active in various milk products. The monoglycerides were fully active in pear juice and apple juice in concentrations as low as 1.25 mM, or about 0.03%. It is notable that the virucidal activities of the lipid compounds were better maintained in the juices than in the milk products. Most likely the compounds, particularly fatty acids and alcohols, bind to proteins and/or lipids in the milk, which may partly block their activity [82]. In the last few decades there has been an increase in research and development of antimicrobial agents which could preferably inactivate a number of human pathogens such as bacteria and viruses. The main reasons for this interest are the growing resistance of microorganism to known drugs, the worldwide threat of HIV and a possible pandemic caused by new virulent strains, for example of influenza virus. In addition to specific drugs there is also a need for new microbicides with less-specific actions against pathogenic microorganisms, which are therefore less likely to lead to the emergence of resistant bacterial or viral strains. For example, the drugs presently used against RSV and other viruses causing respiratory infections are only recommended for high-risk children or severely ill infants, due to efficacy and safety concerns [86, 87]. Therefore, new prophylactic or therapeutic compounds for general use against respiratory viruses would be desirable. The significant virucidal activities of fatty alcohols and lipids on RSV and HPIV2 raise the question of the feasibility of using the most active compounds as ingredients in pharmaceutical formulations against respiratory infections, or as food additives to protect the oral, nasal and respiratory mucosa of infants against viral infection. In relation to a possible use of fatty acids as active antiviral ingredients, Loftsson et al. [88] studied the enhancement of fatty acid extract (FAE) from cod-liver oil on transdermal delivery of acyclovir, which is an HSV-specific antiviral drug, and the activity of the exctract against HSV-1. A 1% FAE caused a 50 000-fold or greater reduction in HSV-1 after 10 minutes, while an ointment containing 30% FAE inactivated the virus about 1.5 millionfold. Also, transdermal studies with variable ointments containing acyclovir showed that the largest acyclovir flux through the skin of hairless mice was obtained from a cream vehicle containing 30% FAE and about 30% propylene glycol. These findings suggest that FAE from cod-liver oil not only enhances skin penetration but might possibly also enhance the antiviral activity of the formulation. Regarding safety concerns, lipids have been shown to be toxic to cell monolayers in cell cultures [30], although antimicrobial formulations containing monocaprin have been found to be harmless to skin and mucosa. Thus, hydrogels have been formulated which contain monocaprin at the highly virucidal concentration of 20 mM (0.5%) [89]. They did not cause

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irritation in the vaginal mucosa of mice and rabbits [10,90], in the skin of hairless mice [90] or in the vaginal mucosa of healthy human volunteers [91], even though they were toxic in cell culture. It is also noteworthy that monoglycerides of fatty acids are classified as GRAS (generally recognized as safe) by the United States Food and Drug Administration and are approved as food additives by the EU (E471). 3.3.4

Antiviral Mechanism of Lipids

Several attempts have been made to elucidate the mechanism behind the antiviral effects of fatty acids and monoglycerides. Sedimentation analysis of bacteriophage
6 inactivated by lipids showed that treatment with 10 ␮g/ml oleic acid for 30 minutes resulted in the loss of the viral envelope, whereas 50 ␮g/ml caused a complete disruption of the virus particle [76]. Another study, investigating the viral particles of mouse mammary tumour virus after inactivation by the cream fraction of human milk, also showed degradation of the viral envelope [70]. In yet another report [74] the effect of fatty acids on VSV particles was studied using negative staining and electron microscopy (EM). It was demonstrated that 0.5 mg/ml of linoleic acid partly disrupted the viral envelope and that it was completely disintegrated by 1 mg/ml of this particular fatty acid (Figure 3.3). The effect of human milk and linoleic acid on Vero cells was analysed by studies of the cells with SEM [74]. The cell monolayers were completely disintegrated by linoleic acid, as well as by antiviral milk samples. As mentioned earlier, micromolar concentrations of linoleic acid reduced the infectivity of enveloped viruses without showing toxicity to animal cells [80]. Further studies by Kohn and coworkers showed that the loss of infectivity was due to disruption of the lipoprotein envelope of these virions, as observed in an EM. Thus, treatment of Sendai and Sindbis viruses with 10 ␮g/ml of linoleic acid for five to eight minutes caused damage or destruction of the viral envelopes. Kohn and coworkers further concluded that since naked viruses lacking host cell-derived envelope are unaffected by unsaturated fatty acids, the envelope must be the target of the virucidal activities of fatty acids. They suggested that the effect of polyunsaturated fatty acids on enveloped viruses is due to a decrease in the rigidity of the biological membranes, which can destroy the viruses by dissolving their envelopes [80,81]. Hilmarsson et al. [83] found a distinct difference in the activities of medium-chain monoglycerides against HSV, monocaprylin and monomyristin being markedly less effective than monocaprin and monolaurin upon contact for one minute. To further address this matter, a series of monoglycerides with chain lengths ranging from 8 to 12 carbons were tested in 10 mM concentration in one minute against HSV-1. Both monocaprin (10 : 0) and nonanoic acid monoglyceride (9 : 0) were fully active and reduced the virus titre 100 000-fold, while monocaprylin (8 : 0), undecanoic acid monoglyceride (11 : 0) and monolaurin (12 : 0) showed about 10 000-fold reduction. Thus, monoglycerides with 9- or 10-carbon chain length were highly virucidal in 10 mM concentration against HSV-1 in a short time, and addition or reduction of one carbon in the chain length seemed to reduce their virucidal activities [unpublished data]. Thus, a direct comparison of the virucidal activities of a series of saturated monoglycerides with increasing chain lengths suggests a possible relationship between the chain length and the thickness of the viral envelope. A difference in glyceride solubility is a less likely explanation of the difference in virucidal activity, since both monocaprylin (8 : 0), with the shortest chain length and highest solubility, and monolaurin

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(b)

(a)

(c)

Figure 3.3 Electron micrographs of negatively stained particles of vesicular stomatitis virus incubated at 37 ◦ C for 30 minutes in (a) maintenance medium, showing normal intact particles covered with spikes, (b) 0.5 mg/ml linoleic acid, showing that the viral envelopes are no longer intact, allowing penetration of stain into most particles, and (c) 1 mg/ml linoleic acid, showing virus particles in various stages of disintegration. Bar, 0.1 µm. (Reprinted from [74] with permission from American Society for Microbiology.)

(12 : 0), with the longest chain length and lowest solubility, were less active than monocaprin (10 : 0). Exactly how lipids inactivate viruses is unknown, but it has been shown that fatty acids cause disintegration of the bilayer lipid envelope [70, 74, 76, 80, 81]. Medium- and longchain alcohols may act in a similar way; that is, penetrating the envelope of the virus by

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hydrophobic effect, making it permeable to small molecules and thus inactivating the virus. The greater activities of 10- and 12-carbon saturated alcohols and lipids compared with other chain lengths, the higher activities of long-chain unsaturated versus saturated lipids, and the generally higher activities of monoglycerides than the corresponding fatty acids at low concentrations are all notable and have been found for both viruses and bacteria [20,74,75]. Possibly the different activities can be explained by a difference in the degree of penetration into lipid membranes due to the chain length of a lipid compared with the thickness of the membrane, as suggested by the effect of 8- to 12-carbon monoglycerides on HSV. A different antiviral effect has been described for fatty acids, particularly lauric acid, against two enveloped viruses, Junin virus and VSV. In both viruses, micromolar concentrations of lauric acid significantly reduced the virus yield by altering the virus production. This was apparently due to stimulation of triacylgycerol production and increased incorporation into the host cell membrane, leading to decreased insertion of viral glycoproteins and inhibition of virus maturation and release [92, 93].

3.4 Antifungal Activities of Fatty Acids and Monoglycerides 3.4.1

In Vitro Studies

Studies of the effect of acids, including acetic acid, on fungi go back to the work of Clark in 1899 [94] on the germination of a number of different moulds such as Aspergillus and Penicillium. Kiesel in 1913 [95] reported studies on the effect of fatty acids and their salts on the development of Aspergillus niger which showed that the activity of saturated fatty acids increased with the length of the carbon chain up to 11 carbon atoms. A few studies published in the early 1930s mostly confirmed his work. Hoffman and others in 1939 [96] studied the fungistatic properties of fatty acids in buffered agar medium seeded with a mixed mould culture, mostly of Aspergillus, Rhizopus and Penicillium spp. obtained from mouldy food products. The agar plates were incubated for 48 hours at 37.5 ◦ C and then observed for mould growth. All of the natural fatty acids containing from 1 to 14 carbon atoms were tested over a pH range of 2 to 8, as well as some unsaturated acids and isomeric forms. All of the saturated acids markedly inhibited mould growth and the activities varied with the pH and the chain length. All acids with chain lengths of 6 carbon atoms or lower were most active at pH <5, whereas the activities of acids with chain lengths of 7 to 12 carbon atoms were highest at pH 7 to 8. The isomeric forms were generally less active than the corresponding natural acids, but unsaturation increased the fungistatic activity of an acid and the effective pH range, as was found for the unsaturated 11-carbon hendecenoic acid (undecylenic acid). Wyss and coworkers in 1945 [97] studied fungistatic and fungicidal actions of fatty acids and related compounds on Trichophyton and Aspergillus. The fungistatic effect was tested by growing the fungus in a medium containing the test compound. The compound concentration which just fully inhibited growth was measured. The fungicidal activity was measured by adding spore suspensions into solutions containing the prospective fungicidal compound. Samples were removed at intervals and tested for surviving spores. The results on the fungistatic actions were in agreement with the findings of previous workers in that the effect of fatty acids on both fungal species increased with the chain length, up to the

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point where solubility became the limiting factor. Unsaturated fatty acids were more active than the corresponding saturated acids. For Aspergillus the optimum chain length was 11 carbon atoms, whereas for the more sensitive Trichophyton spp. it was 13 to 14 carbon atoms. The fungistatic activity of the fatty acids increased with decreasing pH between 4.5 and 7.5, particularly for the short-chain acids. Undecylenic acid was found to be one of the most fungistatic fatty acids for all of the species tested and was most active at pH 5.5 against Trichophyton spp., which are a common cause of skin infections such as athlete’s foot. The fungicidal activity of this unsaturated 11-carbon atom fatty acid was tested against Trichophyton interdigitale at pH 6.5 and 37 ◦ C. The acid, in a concentration of 0.2%, killed 100 000 fungous spores in five minutes, but the killing time increased at lower concentrations. The question of the difference between fungistatic and fungicidal action was addressed by Grunberg [98], who argued that these terms had been used too loosely in the past. According to his distinction, a fungistatic compound is one which inhibits or prevents the growth of fungi, while a fungicidal compound has a much more final effect in that it kills the fungi. Admitting that in some cases it may be difficult to differentiate between the two actions, he nevertheless developed methods designed to distinguish between inhibition and killing of fungi caused by chemotherapeutic agents in vitro. These methods were used to study the action of a series of acids from formic acid (1 : 0) to caprylic acid (8 : 0) against strains of Trichophyton gypseum and Trichophyton purpureum at various pHs. The results were mostly in agreement with previous studies by others, such as that both fungistatic and fungicidal actions of the fatty acids increased with the carbon chain length and decreased with an increase in pH. No appreciable difference was observed in the fungistatic and fungicidal effects of fatty acids against the two different strains of Trichophyton, although infections due to T. purpureum were more resistant to therapy than those caused by T. gypseum. Two important conclusions were drawn from this study, namely that the activity of the fatty acids was due to the undissociated molecules rather than to the ions and that emulsions of the higher fatty acids were more active than solutions at alkaline pH. This was explained by the fact that microorganisms tend to clump around globules of fatty acids in an emulsion, where their concentration is higher than in a solution. Chattaway and his coworkers [99] studied the inhibiting effect on endogenous respiration of Microsporum spp. caused by saturated fatty acids of chain lengths ranging from 2 to 14 carbon atoms. The fatty acids strongly inhibited the respiration and the effect increased with the chain length and with decreasing pH, indicating that the undissociated form was most active. Release of soluble cell constituents from mycelium of Microsporum canis, caused by treatment with a fatty acid, was also measured. It was most rapid at low pH and correlated with the respiratory inhibition. Both effects were closely analogous to those found by Wyss et al. [97] for the fungistatic and fungicidal activity of fatty acids on Trichophyton. Prince in 1959 [100] studied the effect of undecylenic acid and its calcium salt on the growth and respiration of various dermatophytes, such as Trichophyton and Microsporum, at various pHs. The acid and its salt were equally active at pH 4.5 to 6.0. The activity decreased at higher pH, more for the salt than for the acid. This pH effect was in agreement with data from other investigators, who had shown that the antifungal activity of fatty acids and their salts is greatest at low pH, where ionization is repressed. This supports the hypothesis that the undissociated fatty acid is the molecule most responsible for the antifungal effect.

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(b)

Figure 3.4 Effects of capric acid on the ultrastructure of C. albicans as demonstrated by TEM of ultrathin sections of yeast cells. (a) Cell from the control sample with intact cytoplasm, nucleus (N), vacuoles (V) and mitochondria (M). (b) Cell from samples treated with 10 mM capric acid for 30 minutes, demonstrating disorganization of the cytoplasm. No visible changes are seen in the shape or size of the cell wall. (Reprinted from [102] with permission from American Society for Microbiology.)

Candida albicans exhibits two cell morphologies: the round planktonic form, which functions in the spread of the yeast, and the elongated hyphae, which are the virulent form of C. albicans associated with active infection. Recent studies [101] have shown that undecylenic acid inhibits the appearance of germ tubes, which are the first hyphal formation emerging from a planktonic cell, at concentrations that do not inhibit cell proliferation. The acid therefore showed a specific inhibiting action on the morphogenesis of C. albicans, possibly through inhibition of enzymes involved in lipid metabolism. Bergsson et al. [102] studied the fungicidal effect of fatty acids and monoglycerides on the planktonic form of C. albicans and showed that 10 mM capric acid caused a more than 6 log10 reduction in viable cell number in 10 minutes. Lauric acid in lower concentrations also killed the yeast cells in large numbers after contact for 30 minutes. An EM examination of cells of C. albicans killed by capric acid showed disorganization of the cytoplasm, whereas no changes were seen in the shape of the cell wall (Figure 3.4). Undecylenic acid was not tested in this study. In the late 1930s and the 1940s there was considerable interest in the antimycotic activity of fatty acids and related compounds, particularly in connection with treatment of dermatophytoses. These are fungal infections caused by dermatophytes, which is a common name for fungi that cause infections of the skin (ringworm), hair and nails in animals and humans, such as species of Trichophyton, Microsporum and Epidermophyton. Thus, at the First Annual Meeting of the Society for Investigative Dermatology held in New York in 1938, Peck and Rosenfeld [103] reported a comprehensive study on the effect of hydrogen ion concentration, fatty acids and vitamin C on the growth of dermatophytes in culture medium, particularly of Trichophyton gypseum, which is a pathogenic fungus causing dermatophytosis in the hair, skin and nails of humans. After determining that the fungus grew well in vitro at pH ranging from 4.0 to 10.0, Peck and Rosenfeld studied the

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fungicidal effect of low-carbon fatty acids, beginning with formic acid with one carbon atom and concluding with capric acid with 10 carbon atoms. All of the fatty acids studied had fungicidal effect at pH above 4.0, but differed in activities. The acids with an odd number of carbon atoms tended to be more fungicidal than acids with even numbers, the most powerful being valeric acid with five carbon atoms. The branched-chain isomers, which are not found in nature, were less fungicidal than the corresponding natural fatty acids and, with one exception, the sodium salts were less fungicidal than the corresponding acids. The unsaturated 11-carbon undecylenic acid, derived from castor-bean oil, was also studied and found to be almost as fungicidal as valeric acid. In contrast to sodium salts of other acids studied, sodium undecylenate was much more active than the acid, possibly due to its increased solubility. The undecylenate was the most fungicidal lipid tested in this study, not only against Trichophyton gypseum, but also against Epidermophyton inguinale and Monilia albicans. It was concluded that there is a definite relationship between structure and fungicidal action of fatty acids and that a double bond is probably important for the activity. It had previously been observed that sweat had fungicidal properties, which seemed to be due to its content of fatty acids. Several of the fungicidal acids were shown to be present in sweat, such as acetic (2 : 0), butyric (4 : 0), caproic (6 : 0), caprylic (8 : 0) and probably capric (10 : 0) acid. In the discussion following his presentation at the meeting, Peck emphasized their possible role in the natural fungicidal action of sweat and the possibility of using some of these acids as therapeutic agents in a more physiological method of treating fungal infections. He proposed an interesting idea for treatment, namely ingestion of foods containing these fatty acids, or even some of the acids themselves, which from a theoretical point of view might increase the content of these substances in sweat and thus create a ‘natural protective mantel’. In a later publication [104], Peck and his coworkers studied the fungicidal effect of human sweat on three dermatophytes, Trichophyton gypseum, Epidermophyton inguinale and Monilia albicans. Small amounts of sweat were collected from the skin of people sitting for an hour under a carbon-filament lamp. Filtered sweat was added to Sabouraud’s bouillon in quantities of 10 to 90%. Fifty per cent and higher concentrations of the heat sweat at pH <7 prevented growth of T. gypseum in the bouillon. Large quantities (275 to 340 ml) of perspiration were also collected from people sitting for hours inside a bag at a temperature of 41 ◦ C. After being alkalinized by addition of sodium hydroxide, this sweat, which was much more dilute than normal sweat, had no activity against T. gypseum. Under normal conditions, evaporation of sweat from the skin tends to concentrate its ingredients. The collected sweat was therefore evaporated to dryness and a series of tubes of Sabouraud’s bouillon containing from 1 to 10% of dry sweat was prepared. A 1% concentration of sweat had no effect on the growth of T. gypseum, whereas 4 to 7% completely inhibited the growth, depending on the sweat samples, which were either from single individuals or pooled. An analysis of two of the samples revealed similar amounts of acetic and propionic acids (3 : 0), whereas there was a considerable difference in the contents of caproic and caprylic acids. The fungicidal action of individal constituents of sweat against T. gypseum was determined in bouillon cultures. Caproic acid was by far the most active in low concentrations, whereas formic, acetic, propionic, butyric and caprylic acids were all equally active, but less so than caproic acid. With the exception of sodium propionate, and to a certain extent

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sodium butyrate, the sodium salts were much less fungicidal than the corresponding acids. The activities of the sodium salts had to be considered because, depending on the pH on the surface of the skin, the sweat might be alkalinized and the acids thus converted into salts. This might make the sweat less fungicidal, although sodium propionate was equally active as the acid. Lactic acid (2-hydroxypropanoic acid), and particularly sodium lactate, were found to be much less fungicidal than the fatty acids and their salts. However, in view of the relatively large concentration of lactic acid in sweat, it might be of practical importance as a fungicide. M. albicans was much more resistant than T. gypseum to any of the fungicides present in sweat, whereas E. inguinale was generally more susceptible than T. gypseum. 3.4.2

In Vivo Studies

Because sodium propionate was found to be equally as fungicidal as its acid, it was tested therapeutically against various types of fungous infection in clinical trials [104]. The preparations tested were an alcoholic solution containing 10% propionate, a powder and an ointment containing 15% propionate. An alcoholic solution containing low amounts of propionic, butyric, caprylic, lactic and ascorbic acids was also used. Application of these preparations, given alone or in combinations, gave satisfactory results and led to either healing or improvement, depending on the condition treated. Tinea cruris (jock itch) responded well, as did other infections caused by E. inguinale and T. gypseum. The solution containing a mixture of compounds seemed to give the best results. Ringworm of the scalp, caused by Microsporum audouini, was not affected by any of the preparations; nor was epidermophytosis of the feet caused by T. purpureum. The authors speculated about the role of sweat as a natural protective film on the skin against fungous infections. It had been suggested by other investigators that the hydrogen ion concentration of the sweat is in itself important in protecting the skin against infections and that the alkaline pH on some areas of the skin could explain their apparent susceptibility to pathogenic fungi. Arguing against this opinion, Peck and his coworkers [104] claimed that the pH effect is due to the fact that the acids in sweat are less fungicidal at alkaline pH and, more importantly, are changed into alkali salts, which are less fungicidal than the free acids. Quoting the work by other investigators who had studied the distribution of sweat on the surface of the body, Peck and his coworkers pointed out that areas such as the axillas, where sweating is profuse, are normally free of fungous infections, which attack the skin outside this area. In the groin, where fungous infections (tinea cruris/jock itch) are more common, there is relatively little sweat. The distribution of sweat on the feet was also considered in relation to fungous infection. There is little actual sweat between the toes and the moisture which is common in this area may cause maceration of the skin but has little fungicidal activity. As is commonly known, this area is an ideal location for fungal growth. From their own results and those of others, Peck and his coworkers suggested that there is a relation between the localization of fungous infection and the distribution of sweat on the surface of the body. Areas with the greatest concentration of sweat seem to be less prone to infection by fungi. According to this view, topical application of fungicidal ingredients of sweat would make sense as a treatment for some of these infections, as is also indicated by the results of the clinical experiments described in the paper. The authors proposed this type of treatment as a physiologic approach to therapy which might cause less irritation and complicating sequelae than treatment with other, less natural fungicidal agents. Thus,

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this form of therapy would simply augment the natural fungicidal action of the sweat. In their paper, the authors again proposed their idea of increasing the fungicidal elements in sweat, either by consumption of certain foods or by administration of fatty acids or their salts in concentrated form, either orally or parenterally. In 1945, Shapiro and Rothman [105] published a paper on the treatment of dermatomycoses (dermatophytoses) with undecylenate ointment containing 20% zinc undecylenate and 5% undecylenic acid. In a group of 113 patients with dermatomycosis of the feet, 86% achieved complete clinical cure within four weeks of treatment regardless of the species of causative fungi, which included Trichophyton, Epidermophyton and Monilia. This was slightly superior to older standard preparations used in treatment of this condition, but in contrast to them the undecylenate ointment was nonirritating. A rapid clinical cure with a complete absence of irritation of the skin was also observed in patients with tinea cruris and tinea axillaris. Treatment of ringworm of the body (tinea corporis) and the scalp (tinea capitis) was less successful. In general, treatment of intertriginous skin had a more favorable effect compared with treatment of skin of nonintertriginous regions. The fact that the undecylenate ointment was strongly fungicidal and at the same time nonirritating to the skin was considered its main virtue. Based on previous in vitro studies, which showed that sodium caprylate is particularly effective against Monilia albicans (Candida albicans), Keeney in 1946 [106] treated moniliasis (candidiasis) of the skin and mucous membranes with preparations of this fatty acid sodium salt. In this clinical study, children with oral thrush were successfully treated with a 20% aqueous solution of caprylate. An adult female patient was treated with 10% caprylate ointment for extensive cutaneous moniliasis and with 10% caprylate vaginal jelly and talcum for vaginal moniliasis. Both treatments were effective but the vaginal preparations caused irritation, particularly the jelly. Neuhauser and Gustus in 1954 [107] addressed the problem of the increased number of cases with intestinal infections due to Candida albicans. Large numbers of this organism appeared in the faeces of patients treated with antibiotics, because of a change in the normal intestinal flora caused by these antibacterial drugs. Although they most often disappeared upon discontinuation of antibiotic therapy, the fungous infections became chronic in some cases, with more or less severe symptoms and large numbers of C. albicans in the faeces, and sometimes with fatal outcome. Because oral administration of undecylenic acid had recently been successfully used to prevent monilial complications caused by the use of antibiotics, Neuhauser and Gustus decided to attempt to treat a seriously ill patient with a saturated complex of undecylenic acid and an anion-exchange resin. Such resins, in the form of a powder, had been successfully used for a number of years as carriers in the administration of gastrointestinal drugs, releasing the drug continuously at a controllable rate. Before giving the complex to the patient, each of the authors took large doses of the powdered product by mouth without experiencing any ill effects. The patient recovered fully from the intestinal moniliasis after being treated over a period of time. Generalized moniliasis of the skin was successfully treated at the same time with an ointment consisting of 10% calcium propionate and 10% sodium propionate. In order to determine whether other fatty acid–resin complexes were active against C. albicans, in vitro tests on Sabourauds’s agar were carried out. Of all the fatty acid–resins tested, the caprylic acid–resin complex was found to be the most active in preventing the growth of C. albicans on agar medium. It was markedly more active than the undecylenic acid–resin, both at acid and at slightly alkaline

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pH. Treatment of several patients with caprylic acid–resin complex resulted in complete recovery from intestinal moniliasis without any undesired side effects. The recovery was more rapid than following treatment with undecylenic acid–resin. Rothman and coworkers in 1946 and 1947 [108,109] studied ringworm of the scalp hair, which was endemic among school children in the United States. This condition, which is caused by the fungus Microsporon audouini, clears up spontaneously during puberty and the scalps of adults are not susceptible to it. From clinical observation it had been known that under the influence of sex hormones during puberty the sebacious glands and their secretion changed profoundly, and there was a greatly increased amount of sebum on the scalp of adults, with a change of odour. Hypothesizing that the spontaneous cure of ringworm of the scalp during puberty was due to changes in the chemical composition of sebum, the fungicidal properties of adult hair fat were investigated and compared with those of hair fat from children. In the beginning, hair sweepings were collected from neighborhood barber shops, excluding children’s hair. Ether extracts of the adult human hair were found to contain lipids which inhibited the growth of M. audouini on culture media, but were 10 times less active against Trichophyton and other pathogenic fungi which infect the hair of adults. The fungistatic action of lipids extracted from the hair of children under 11 years of age was more than five times less than that of adult lipids, as tested for prevention of M. audouini growth on Sabouraud’s agar. A preliminary fractionation of the lipids showed that the free fatty acid components had the strongest inhibitory activity, and that a small fraction containing short-chain saturated fatty acids was the most active and prevented the growth of M. audouini on the agar medium in concentrations of 0.0002 to 0.0005%. It was shown that this threshold concentration of the fatty acids, which completely inhibits the growth of fungi, was likewise fungicidal. With a possible therapeutic treatment in mind, Rothman and coworkers collected 45 kg of adult hair from more than 10 000 haircuts in large downtown barber shops. An ether extraction yielded 230 grams of free fatty acids, of which acids with chain lengths of 7, 9, 11 and 13 carbon atoms were 2.2 grams or about 1%. The 9-carbon pelargonic acid was prominent in this most active fraction. The more abundant, higher-molecular-weight fractions consisted of straight-chain saturated and unsaturated fatty acids with up to 22 carbon atoms, either odd- or even-numbered. The authors stated that human hair fat was, at that time, the only known natural source of straight-chained fatty acids with odd numbers of carbon atoms, with the exception of formic and propionic acids. However, odd-numbered acids with branched chains had previously been found in wool fat. As shown by Peck et al. [103], acids containing odd numbers of carbon atoms are more fungistatic than evennumbered acids, and this was strongly evident in the high activity against M. audouini of acids with 7 to 13 carbon atoms. Therapeutic trials on children with ringworm of the scalp were carried out in which total hair fat, the most active fatty acid fractions, and pelargonic acid and its equally fungicidal sodium salt were all applied to the scalp by various methods. No therapeutic effect was obtained. This was shown to be due to the fact that neither fatty acids nor their salts were able to penetrate the keratin and therefore they could not reach the fungal spores inside the hair. However, it was suggested that local treatment of exposed but noninfected children might prevent the spread of this fungal disease in crowded facilites, such as in schools and orphanages. The glabrous skin of adults is not immune to M. audouini infections and adults who take care of children with ringworm of the scalp are exposed to the infection on

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other parts of their body. Apparently, the concentration of the active acids on the glabrous skin is below the critical level needed to protect against M. audouini, possibly because the sebacious glands are smaller there than on the scalp. The most notable result of this study was the finding that with onset of puberty the sebaceous glands of the scalp begin to secrete a sebum containing an increased amount of highly fungistatic and fungicidal fatty acids with a selective activity against M. audouini, which makes adults naturally immune to infections by this fungus. This adult type of fat diffuses into the follicular canals and prevents infection of new hairs, which grow out following the shedding of old infected hairs. Consequently, this natural cure in puberty is a very slow process. This process is an example of the role of lipids in the natural defence against infections. Normal saturated fatty acids are thus fungicidal agents used by nature. Although only about 2% of the total fatty acid contents of the hair had a significant role in the immunity of adults to M. audouini, the remaining 98% were considered to be important in the physiology of the sebacious gland secretion, particularly in view of its complex mechanism and great number of specific products. Following the study of Rothman and his coworkers, Perlman in 1949 [110] attempted treatment of children with ringworm of the scalp by oral administration of undecylenic acid in doses of 1 gram given three times daily. The treatment was unsuccessful and was discontinued. On the other hand, patients with chronic psoriasis and neurodermatis showed improvements after treatment with undecylenic acid for varying periods of time, with disappearance of lesions and relief of itching. The dose of 5 grams given three times daily caused no harmful effects but only some unpleasant side effects. Between 1977 and 1983, several studies were carried out to evaluate the efficacy of undecylenic acid preparations in topical treatment of tinea pedis (athlete’s foot) and of dermatophytoses of the glabrous skin and groin [111–115]. Treatments with ointments and powders containing either undecylenic acid or zinc undecylenate were found to be safe and effective and comparable to a common fungicidal cream. Desinex cream and a number of other preparations containing undecylenic acid as the active ingredient are on the market as over-the-counter antifungals [116].

3.5

Conclusions

In vitro studies have shown that microbes vary greatly in their susceptibility to microbicidal lipids: some are killed in a short time at low lipid concentration, others in a longer time or at higher concentrations. Which lipids are the most active against each bacterium, virus or fungus is also variable. Unsaturated fatty acids and medium-chain saturated fatty acids and their monoglycerides are generally most active against pathogenic bacteria and viruses and kill them rapidly and in large amounts on contact, whereas low- to medium-carbon fatty acids and their salts are in many cases active against fungi. The microbicidal action of lipids is affected by pH, the activity generally being higher in acid environment than at neutral or alkaline pH. Several studies suggest that lipids, especially unsaturated fatty acids, play a role in the natural defence against bacterial infections in skin and mucosal membranes and that short- and medium-chain fatty acids in sweat are protective against fungal infections. Although a few attempts have been made to treat bacterial and viral infections in skin and

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mucosa with pharmaceutical formulations containing microbicidal lipids it has not yet been shown that such formulations are helpful in treatment or prevention of bacterial and viral infections in animals or humans. On the other hand, experiments have shown that treatment with formulations containing medium-chain fatty acids, particularly undecylenic acid and salts thereof, has a therapeutic effect in several types of fungal infection. Pharmaceutical dosage forms, such as creams, ointments and powders, are available as over-the-counter antifungals.

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86. Simoes, E.A. (2002) Immunoprophylaxis of respiratory syncytial virus: global experience. Resp Res., 3, 26–33. 87. Wyde, P.R., Chetty, S.N., Timmerman, P. et al. (2003) Short duration aerosols of JNJ 2408068 (R170591) administrated prophylactically or therapeutically protect cotton rats from experimental respiratory syncytial virus infection. Antiviral Res., 60, 221–231. ´ 88. Loftsson, T., Thormar, H., Olafsson, J.H. et al. (1998) Fatty acid extracts from cod-liver oil: activity against herpes simplex virus and enhancment of transdermal delivery of acyclovir in vitro. Pharm. Pharmacol. Commun., 4, 287–291. ´ 89. Kristmundsd´ottir, T., Arnad´ ottir, S.G., Bergsson, G. and Thormar, H. (1999) Development and evaluation of microbicidal hydrogels containing monoglyceride as the active ingredient. J. Pharmaceut. Sci., 88, 1011–1015. 90. Neyts, J., Kristmundsd´ottir, T., De Clercq, E. and Thormar, H. (2000) Hydrogels containing monocaprin prevent intravaginal and intracutaneous infections with HSV-2 in mice: impact on the search for vaginal microbicides. J. Med. Virol., 61, 107–110. 91. Arnfinnsd´ottir, A.V., Geirsson, R.T., Hilmarsd´ottir, I. et al. (2004) Effects of monocaprin hydrogel on the vaginal mucosa and bacterial colonisation, 34th Congress of Nordic Federation of Societies of Obstetrics and Gynecology, Helsinki, Finland, p. 91. 92. Hornung, B., Amtmann, E. and Sauer, G. (1994) Lauric acid inhibits the maturation of vesicular stomatitis virus. J. Gen. Virol., 75, 353–361. 93. Bartolotta, S., Garc´ıa, C.C., Candura, N.A. and Damonte, E.B. (2001) Effects of fatty acids on arenavirus replicaton: inhibition of virus production by lauric acid. Arch. Virol., 146, 777–790. 94. Clark, J.R. (1899) On the toxic effect of deleterious agents on germination and development of certain filamentous fungi. Bot. Gaz., 28, 289–327. 95. Kiesel, A. (1913) Recherches sur l’action de divers acides et sels acides sur le d´eveloppement de l’aspergillus niger. Ann. de l’Inst. Pasteur, 27, 391–420 96. Hoffman, C., Schweitzer, T.R. and Dalby, G. (1939) Fungistatic properties of the fatty acids and possible biochemical significance. Food Res., 6, 539–545. 97. Wyss, O., Ludwig, B.C. and Joiner, R.R. (1945) The fungistatic and fungicidal action of fatty acids and related compounds. Arch. Biochem., 7, 415–425. 98. Grunberg, H. (1947) The fungistatic and fungicidal effects of the fatty acids on species of Trichophyton. Yale J. Biol. Med., 19, 855–876 99. Chattaway, F.W. and Thompson, C.C. (1956) The action of inhibitors on dermatophytes. Biochem. J., 63, 648–656. 100. Prince, H.N. (1959) Effect of pH on the antifungal activity of undecylenic acid and its calcium salt. J. Bacteriol., 78, 788–791. 101. McLain, N., Ascanio, R., Baker, C. et al. (2000) Undecylenic acid inhibits morphogenesis of Candida albicans. Antimicrob. Agents Chemother., 44, 2873–2875. ´ Thormar, H. (2001) In vitro killing of Candida 102. Bergsson, G., Arnfinnsson, J., Steingr´ımsson, O., albicans by fatty acids and monoglycerides. Antimicrob. Agents Chemother., 45, 3209–3212. 103. Peck, S.M. and Rosenfeld, H. (1938) The effects of hydrogen ion concentration, fatty acids and vitamin C on the growth of fungi. J. Invest. Dermatol., 1, 237–265. 104. Peck, S.M., Rosenfeld, H., Leifer, W. and Bierman, W. (1939) Role of sweat as a fungicide. With special reference to the use of constituents of sweat in the therapy of fungous infections. Arch. Dermatol. Syphilol., 39, 126–148. 105. Shapiro, A.L. and Rothman, S. (1945) Undecylenic acid in the treatment of dermatocytosis. Arch. Dermatol. Syphilol., 52, 166–171. 106. Keeney, E.L. (1946) Sodium caprylate. A new and effective treatment for moniliasis of the skin and mucous membranes. Bull. Johns Hopkins Univ. Hosp., 78, 333–339. 107. Neuhauser, I. and Gustus, E.L. (1954) Successful treatment of intestinal moniliasis with fatty acid–resin complex. Arch. Intern. Med., 93, 53–60. 108. Rothman, S., Smiljanic, A.M. and Weitkamp, A.W. (1946) Mechanism of spontaneous cure in puberty of ringworm of the scalp. Science, 104, 201–203. 109. Rothman, S., Smiljanic, A., Shapiro, A.L. and Weitkamp, A.W. (1947) The spontaneous cure of tinea capitis in puberty. J. Invest. Dermatol., 8, 81–98.

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110. Perlman, H.H. (1949) Undecylenic acid given orally in psoriasis and neurodermatitis. J. Am. Med. Assoc., 139, 444–447. 111. Smith, E.B., Powell, R.P., Graham, J.L., and Ulrich, J.A. (1977) Topical undecylenic acid in tinea pedis: a new look. Int. J. Dermatol., 16, 52–56. 112. Tschen, E.H., Becker, L.E., Ulrich, J.A. et al. (1979) Comparison of over-the-counter agents for tinea pedis. Cutis, 23, 696–698. 113. Chretien, J.H., Esswein, J.G., Sharpe, L.M. et al. (1980) Efficacy of undecylenic acid-zink undecylenate powder in culture positive tinea pedis. Int. J. Dermatol., 19, 51–54. 114. Fuerst, J.F., Cox, G.F., Weaver, S.M. and Duncan, W.C. (1980) Comparison between undecylenic acid and tolnaftate in the treatment of tinea pedis. Cutis, 25, 544–546, 549. 115. Battistini, F., Cordero, C., Urcuyo, F.G. et al. (1983) The treatment of dermatophytoses of the glabrous skin: a comparison of undecylenic acid and its salt versus tolnaftate. Int. J. Dermatol., 22, 388–389. 116. Anon. (2002) Undecylenic acid. Alt. Med. Rev., 7, 68–70.

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4 Antimicrobial Lipids in Milk Charles E. Isaacs Department of Developmental Biochemistry, New York State Institute for Basic Research in Developmental Disabilities, NY, USA

4.1 4.2

4.3

4.4

4.5

Introduction Occurrence 4.2.1 Biosynthesis 4.2.2 Quantitative Assays 4.2.2.1 Measurement of Antibacterial Activity 4.2.2.2 Measurement of Antiviral Activity Molecular Properties 4.3.1 Lipids in Milk and Plant Oils 4.3.2 Dependence of Antimicrobial Activity on Chain Length, Saturation and pH Antimicrobial Activity 4.4.1 Mechanism of Action 4.4.2 Synergy between Antimicrobial Lipids and Antimicrobial Peptides 4.4.2.1 Antimicrobial Peptides and Proteins 4.4.2.2 Potential Synergism between Antimicrobial Milk Lipids and Peptides 4.4.3 Influencing Factors 4.4.4 Spectrum Applications 4.5.1 Additive Advantage to Foods and Biological Products

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4.5.2 Physiological Advantage 4.5.3 Sexually Transmitted Pathogens and Topical Activity Safety, Tolerance and Efficacy Conclusions

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4.1 Introduction Milk is not only a vehicle which delivers nutrients to the newborn, but also a natural functional food. It transports molecules and cells that are part of the secretory immune system and plays an important role in innate defence by protecting mucosal surfaces from infection [1]. Milk lipids are a class of molecule that have broad-spectrum antimicrobial activity in addition to their nutritional value. The antimicrobial activity of lipids, in particular fatty acids, has been studied since the late nineteenth century [2, 3]. Lipid-dependent antimicrobial activity delivered in milk to the neonate results from the release of antimicrobial fatty acids and monoglycerides from milk triglycerides. These triglycerides are present in milk fat globules and represent 98% of the milk fat [4–6]. Monoglycerides, diglycerides and ether lipids are present in trace amounts. The triglyceride core of the milk fat globule is surrounded by a membrane of polar lipids, which prevents hydrolysis of the triglycerides by lipases in milk [7], and therefore human milk lipid-dependent antimicrobial activity is released by lipases in the infant’s gastrointestinal tract [8]. The same is true for bovine milk. The digestion products of bovine milk triglycerides and membrane lipids inactivate Escherichia coli 0157 : H7, Salmonella enteritidis, Campylobacter jejuni, Listeria monocytogenes and Clostridium perfringens [9–12]. Milk lipids are not unique in possessing antimicrobial activity. Human epidermis-derived skin lipids, especially free fatty acids, have been found to inactivate bacterial pathogens, for example Staphylococcus aureus [13–17]. Studies at other mucosal surfaces showed that lung surfactant from humans, dogs, rats and guinea pigs contained free fatty acids that were bactericidal for pneumococci [18–20]. Inhaled pneumococci were killed extracellularly in rats [19] and the antibacterial activity resided in long-chain polyunsaturated free fatty acids in lung surfactant. Fractionation of porcine intestinal lipids showed that antibacterial activity against Clostridium welchii was primarily due to the activity of long-chain unsaturated fatty acids [21]. Free fatty acids isolated from nine species of brown algae [22] inactivated Gram-positive and Gram-negative bacteria. In another study examining Caribbean marine algae [23], over 70% of the lipid extracts from approximately 100 algae tested had antibacterial activity. Fatty acids and their antimicrobial derivatives not only have a protective role in milk and at mucosal surfaces of vertebrates but also appear to be utilized by simpler eukaryotes for antibacterial activity. Lipids are one of a number of ‘nonspecific’ protective factors present in milk which function at mucosal surfaces [24, 25]. These